Theory and practice of prion protein analysis in food products

A. Yu. Prosekov

ISSN 2308-4057. Foods and Raw Materials Vol. 2, No. 2, 2014

THEORY AND PRACTICE OF PRION PROTEIN ANALYSIS

IN FOOD PRODUCTS

A. Yu. Prosekov

Kemerovo Institute of Food Science and Technology, bul'v. Stroitelei 47, Kemerovo, 650056 Russia phone: +7 (923) 606-33-73, e-mail: [email protected]

(Received March 3, 2014; Accepted in revised form April 14, 2014)

Abstract: The article presents the results of the research on methods of identification and quantitative determination of prion proteins in biological samples and multicomponent mixtures based on them. Analysis of nucleotide sequence of DNA encoding the PRNP gene of the prion protein, including phylogenetic and comparative analysis of nucleotide sequences of normal and pathogenic prion protein in cattle, was performed. Oligonucleotide primers for amplification of the PRNP gene of pathogenic prion protein were designed and synthesized. The high specificity of the developed test system was confirmed.

Keywords: prion, protein, encephalopathy, safety, quality, PCR, analysis

UDC 641:577.112 DOI 10.12737/5467

INTRODUCTION

Prions (proteinaceous infectious particles) are a special class of purely protein agents, free of nucleic acids, causing severe diseases of central nervous system in human and a number of higher animals [1-3].

Prion protein can exist in two forms: a noninfectious vitally important protein present in the organism of mammals, including human, and an infectious protein, which is a mutation of the normal prion protein causing prion diseases of animals and man.

Prion diseases are a group of transmissive neurodegenerative diseases of animals and humans. The diseases are characterized by prolonged incubation periods, but rapid progression from the moment of clinical onset of the disease. All prion diseases are lethal and there is no efficient methods of treatment so far. In 1997, Stenly B. Prusiner won the Nobel Prize for the outstanding discovery of prions.

Spongiform encephalopathy in cattle was registered in Great Britain, Switzerland, Ireland, Portugal, France, Germany, the Netherlands, Italy, Denmark, and Falkland Islands. The reported cases of disease were caused by the import of infectious animals or diseased meat-and-bone meal tankage produced from the killing products and used for breeding of the young stock in these countries [4, 5].

Prophylaxis of prion diseases is based on prohibition of the infected meat products or other killing products on food market. In this connection, in the Enactment of the Chief State Medical Officer of the Russian Federation no. 15 of 15.12.2000 «On the Measures for Prevention of Creutzfeldt-Jacobs Disease Spreading on the Territory of the Russian Federation», preventing measures aimed at prohibition of import of diseased meat and meat products were defined for the first time.

Taking this into account, improvement and development of new methods for identification of prion proteins in biological material is of scientific and practical interest.

OBJECTS AND METHODS OF THE STUDY

Whole milk, whole beef blood, blood plasma, cheese, beef muscle tissue, stromal fractions, gelatin, and samples of cattle meat were used. Samples of meat and blood were collected from animals having passed the veterinary control; the carcasses were proven fit for human consumption. The following nucleotide sequences corresponding to the PRNP gene of the prion protein deposited in the GenBank database were analyzed: Equus caballus (house horse), Equus asinus (house donkey), Sus scrofa (pig), Bos taurus (cow), Bos javanicus (Javan bull), Bubalus bubalis (buffalo), Syncerus caffer caffer (African buffalo), Capra hircus (goat), Ammotragus lervia (jubate sheep), Ovis aries (urial), Rangifer tarandus granti (northern deer), Capreolus capreolus (roedeer), Alces alces alces (elk), Cervus elaphus nelsoni (northamerican elk), Cervus dama (fallow deer), and Homo sapiens (human).

In the work, we used standard, common, and original methods, including the phylogenetic analysis of the protein gene nucleotide sequences, differential amplification of specific sequences and real-time polymerase chain reaction (PCR). The experiments involving PCR were performed following the requirements on determination of pathogenic microorganisms in cattle processing products.

Prior to the studies, independently of the analysis method, primary treatment of the samples was performed. In the case of analysis of soft and easily grinded materials (meat, cheese, etc.), averaged sample of the product weighing 1 g was collected, grinded using a sterile scalpel, scissors, and disposable spatula, and homogenized using a porcelain pistil in a ceramic mortar, with thorough mixing of the content.

For samples of dry particulate materials (gelatin) and liquid or semi-liquid materials (milk, blood, etc.), which require no grinding and are homogeneous, disposable spatula or a pipette was used to introduce 100-150 ^L of bulk volume of a sample to an

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Eppendorf tube (5-7 mm from the tube bottom). To prevent cross-contamination, the grinding instruments were used once, washed carefully, and sterilized.

For isolation of different protein fractions of animal origin, the samples were pretreated as follows: muscles of different animals were thoroughly freed from fat and connective tissue; weighed amount (3-4 g) of the tissue was cut by a knife on a watch glass. Distilled water was added at the ratio of 1 : 6 (by mass) and extraction was performed on cold at 0°C for 30 min. Then, the sediment was separated by centrifugation at 83 s-1 for 5 min. The supernatant was carefully decanted and used for quantitative protein determination.

Liquid samples were prepared by dilution in distilled water, so that the protein content in a gel pocket would not exceed 5 ^g per 20 ^L solution.

Determination of the total protein in samples was performed according to a technique of total and protein nitrogen fractions determination in meat, meat products, and protein-containing food products by the burning method of Duma [6].

Protein identification was performed by fingerprinting of peptide masses. Proteins were identified by the mass spectrum of amino acid sequences upon hydrolysis with trypsin in polyacrylamide gel.

To perform the mass spectrometry analysis, 0.5-2 ^L of sample solution and 0.3 of a 20 mg/mL 2,5-dihydroxybenzoic acid solution in 20% acetonitrile aqueous solution with 0.5% TFA (Aldrich) were mixed on a ground steel support. Mass spectra were recorded on an Ultraflex II (Bruker, Germany) tandem MALDI time-of-flight mass spectrometer in the mass range of 700-4500 m/z under laser power optimal for the best resolution and registration of trypsin autolysis peaks, which were further used for internal calibration.

Mass spectra were processed with a FlexAnalysis 2.4 (Bruker Daltonics, Germany) software. If needed, fragmentation spectra of individual peptides were registered under tandem mode. Possible amino acid sequences were indexed in successfully fragmented peptides.

The accuracy of the average [MH+] measured mass in the linear mode was 5 Da. The accuracy of the monoisotopic measured masses in the reflecto-mode without internal calibration was 0.01% and after an additional calibration using trypsin autolysis peaks, 0.005%. Accuracy of the monoisotopic measured masses of fragments was 1 Da.

Molecular mass distribution of the proteins in the samples was evaluated by protein electrophoresis according to Laemmli [7].

Proteins were separated in denaturing 12% separating and 4% concentrating polyacrylamide gel supplemented with 0.1% sodium dodecyl sulfate. Electrophoresis was performed in a separating buffer supplemented with 0.1% sodium dodecyl sulfate under 15 mA. Gel was stained with 0.2% Coomasie Brilliant Blue R250 dye, prepared using glacial acetic acid, at high temperature for 7-10 min and then washed three times with distilled water.

Gels were viewed and imaged using a TCP-20M (Vilber Lourmat, United States) UV-transilluminator at the wavelength of 312 nm. Data storage and processing were performed with a DOC-it-LS gel-documenting system.

Gel calibration was performed using a set of protein markers by SibEnzyme containing 12 highly purified recombinant proteins of molecular mass between 10 and 250 kDa. For quantitative evaluation of normal prion protein content, gel was calibrated using human serum albumin protein solutions of known concentration.

Protein concentration in a sample was calculated according to the formula:

C = (Cp • Cf)/100,

where Cp is the mass fraction of the total protein in a sample, g/100 g, and Sf is the mass fraction of a protein fraction to the total protein content in a samples, g/100 g protein.

In the course of the study, 17 nucleotide sequences of the PRNP prion protein gene deposited in the GenBank were used. To elucidate the differences and search for homologous sequences, NCBI database was used [8, 9]. Nucleotide acid sequences were aligned using the ClustalW software.

For comparative analysis of the DNA nucleotide sequences encoding the PRNP gene, OligoCalc software was used. Phylogenetic tree was designed using the ClustalW software.

Computer-based primer selection analysis for amplification of specific sequences of the pathogenic prion protein was performed using the following software: NCBI Blast2 for determination of homology upon sequencing of relevant primers and Primer3 Output for selection and evaluation of the primers.

Immuno-PCR was performed using the reaction mixtures presented in Table 1.

Table 1. Composition of the PCR reaction mixture

Component Final concentration Component content per 25 ^L of the mixture

10x PCR buffer 0.1 ^M 2.5 ^L

10 mM dNTP mixture 0.2 mM 0.5 ^L

Primer 1 (50 ^L) 1 ^M 0.5 ^L

Primer 2 (50 ^L) 1 ^M 0.5 ^L

Taq DNA polymerase 1.25 un. 0.25 ^L

25 mM MgCl2 1.5 mM 1 ^L

DNA template 0.1-1 ^g Varies in function of concentration in a sample

Deionized water - Adjusted to 25 ^L

RESULTS AND DISCUSSION

Total protein content in the samples is reported in Table 2.

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Table 2. Total protein content in the samples

Subject of the study Mass, mg Total nitrogen content, % Coefficient for calculations Total protein, % Measurement error, ±5,%

Whole milk 195.60 0.656 4.64 3.02 0.31

192.40 0.648

Gelatin 188.12 15.123 5.55 84.32 0.89

180.72 15.538

Whole blood 214.50 3.462 6.25 21.97 1.32

254.80 3.571

Cheese 189.70 4.686 4.64 21.46 0.95

229.60 4.653

Beef 126.50 3.222 5.62 18.46 1.11

110.00 3.347

Water-soluble beef proteins 97.40 1.148 5.62 6.48 0.41

96.50 1.167

Salt-soluble beef proteins 136.30 1.520 5.62 8.52 0.53

142.25 1.528

Stromal beef proteins 186.80 0.675 5.62 3.78 0.23

171.20 0.679

Based on the data presented in Table 2, one may conclude that the total protein content in samples was 84.32 g/100 g for gelatin, 21.97 g/100 g for blood, 21.46 g/100 g for cheese, and 18.46 g/100 g for beef, with 6.48 g/100 g of water-soluble beef proteins, 8.52 g/100 g salt-soluble beef proteins, and 3.78 g/100 g stromal proteins.

We did not succeed to optimize the conditions for

separation of cattle whole blood electrophoretic separations, therefore, we analyzed the blood plasma which was obtained by centrifugation at 3000 rpm for 5 min. The supernatant containing light fractions of blood proteins was used for analysis. The results of protein fraction distribution in the samples are presented in Fig. 1 and Table 3.

Fig. 1. PAGE in a 12% separating and 4% concentrating gel: M, molecular weight marker; A, beef protein water-soluble fraction; B, beef protein salt-soluble fraction; C, blood plasma; D, gelatin; E, whole milk; F, cheese.

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Table 3. Mass fraction of the total protein and fraction distribution of the proteins

Sample Number of samples Total protein, g/100 g Number of protein fractions in a range

15-30 kDa 30-40 kDa 40-250 kDa

Beef protein water-soluble fraction 20 6.48 1 2 6

Beef protein salt-soluble fraction 20 8.52 0 0 6

Beef protein stromal fraction 20 3.78 0 0 2

Blood plasma 20 9.73 1 2 8

Gelatin 10 84.32 0 0 3

Whole blood 20 3.02 6 0 2

Cheese 20 21.46 4 0 1

In the course of the study, we found that in fractions of stromal and salt-soluble beef proteins there are no low molecular weight protein fractions, which is in good agreement with the literature data, while in the fraction of water soluble proteins there are two protein fractions with masses from 30 to 40 kDa. The indicated protein mass is within the range of normal prion protein mass.

The obtained electrophoresis diagrams of blood plasma samples indicate the presence of two protein fractions with masses from 30 to 40 kDa.

Electrophoresis separation of industrial samples of gelatin, which is produced by partial hydrolysis of

collagen obtained from cattle nails, jacket and skin, strings, and tendons, demonstrated high grade of purity. No low molecular weight protein fractions were observed.

Milk and cheese proteins were also fractionated (Table 4). Analysis of electrophoresis diagrams indicates the presence of traditional milk proteins in all samples. Caseins are characterized by molecular masses of ~22-32 kDa; P-lactoglobulin, ~18 kDa; a-lactalbumin, ~14 kDa; lactoferrin, 80 kDa; and serum albumin, ~66 kDa. No alien protein fractions weighing from 30 to 40 kDa was detected in the samples.

Table 4. Fraction composition of whole milk and cheese proteins

Band number Molecular weight, kDa Protein %, to the total casein content %, to the total serum protein content %, to the total protein content

Е 1 73.82 lactoferrin - 17.76 3.39

Е 2 68.11 blood serum albumin (SA) - 17.04 43.24

Е 3 29.63 as1-casein 53.99 - 23.84

Е 4 27.83 P-casein 29.77 - 4.14

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Е 5 26.09 as2-casein 5.17 - 8.87

Е 6 25.28 к-casein 11.07 - 8.93

Е 7 18.62 P-lactoglobulin - 44.85 4.05

Е 8 15.88 a-lactalbumin - 20.34 3.39

D1 32.11 as1-casein 37.31 - 37.31

D2 30.34 as2-casein 11.23 - 11.23

D3 28.03 P-casein 41.50 - 41.50

D4 26.51 к-casein 9.96 - 9.96

The absence of protein with molecular mass corresponding to that of normal prion protein (30-40 kDa) evidences the low possibility of prion protein presence in samples of whole milk, cheese, and salt-soluble and stromal beef proteins and therefore, the low level of infectiveness of the samples under study. The standard procedure of veterinary control, that is the certificate of fit for human consumption, is sufficient.

Further on, for unambiguous identification of the protein fractions in the above-indicated samples as normal prion proteins, one-dimensional electrophoresis was followed by the protein in-gel cleavage with trypsin and identification by peptide mass fingerprinting.

Samples selected for studies are presented in Table 5.

Table 5. Protein samples for investigation

Sample name Protein mass, kDa

Beef protein water-soluble fraction 32.38

Blood plasma 34.89

Mass spectrum of amino acid sequences upon ingel hydrolysis with trypsin was used for protein identification.

Quantitative content of the normal prion protein in samples was estimated by electrophoresis according to Laemmli followed by staining of the gel with Coomasie Brilliant Blue R250 (Table 6).

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Table 6. Protein fraction composition of samples under study

Sample name Molecular weight, kDa %, to the total protein content %, to the total protein content in a sample Mass fraction, %

Beef protein water-soluble fraction 37.42 18.19 10.78 0.74

32.38 7.41 0.51

Blood plasma 39.17 22.06 21.03 2.05

34.89 1.03 0.1

Relative content of protein fractions from 30 to 40 kDa in fractions of beef water-soluble proteins was 18.19% to the total amount. Electrophoresis diagrams of blood plasma indicate that the relative content of protein fractions from 30 to 40 kDa in blood plasma was 22.06%. Therefore, our study evidences that the fractions of water-soluble proteins from beef and blood plasma under study indeed are normal prion proteins of the cattle.

Phylogenetic connections between the organisms may be elucidated by comparison of sequences of whole genes or their fragments encoding ribosomal RNA. The data on completely or partially sequenced rRNA genes of different organisms are deposited in

international databases and are available in the internet. Today, methods based on determination of ribosomal gene nucleotide sequences are widely used for identification of different infection types [10].

We chose the following PRNP sequences: Equus caballus (house horse), Equus asinus (house donkey), Sus scrofa (pig), Bos taurus (cow), Bos javanicus (Javan bull), Bubalus bubalis (buffalo), Syncerus caffer caffer (african buffalo), Capra hircus (goat), Ammotragus lervia (jubate sheep), Ovis aries (urial), Rangifer tarandus granti (northern deer), Capreolus capreolus (roedeer), Alces alces alces (elk), Cervus elaphus nelsoni (northamerican elk), Cervus dama (fallow deer), and Homo sapiens (human) (see Fig. 2).

gi|27733849 Equus ATGGTGAAAAGCCACGTAGGCGGCTGGATTCTGGTTCTCTTTGTGGCCAC 50 gi| 119514511 Equus ATGGTGAAAAGCCACGTAGGCGGCTGGATTCTGGTTCTCTTTGTGGCCAC 50

gi| 119489983 Sus ATGGTGAAAAGCCACATAGGTGGCTGGATCCTCGTTCTCTTTGTGGCCGC 50

gi| 119489801 Bos ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGCCAT 50

gi|54125480 Bos ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGCCAT 50 gi|54125508 Bubalus ATGGTGAAAAGACACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGTCAT 50 gi|54125464 Syncerus ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGTCAT 50 gi| 119514499 Capra ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGCCAT 50 gi| 119655282 Ammotragus ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGCCAT 50 gi|89160951 Ovis ATGGTGAAAAGCCACATAGGCAGTTGGATCCTGGTTCTCTTTGTGGCCAT 50

gi|73697718 Rangifer ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi|50442265 Rangifer ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi|50442321 Capreolus ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi|50442307 Alces ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi| 158714095 Cervus ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi|50442285 Cervus ATGGTGAAAAGCCACATAGGCAGCTGGATCCTAGTTCTCTTTGTGGCCAT 50 gi|308194928 Homo ATGGCG-AACCTTGGCTGCTGGATGCTGGTTCTCTTTGTGGCCAC 44

gi|27733849 Equus ATGGAGTGACGTGGGGCTCTGCAAGAAGCGACCGAAGCCTG—GAGGAT 97

gi| 119514511 Equus ATGGAGTGACGTGGGGCTCTGCAAGAAGCGACCGAAGCCTG—GAGGAT 97

gi| 119489983 Sus ATGGAGTGACATAGGGCTCTGCAAGAAGCGACCAAAGCCTGGCGGAGGAT 100 gi| 119489801 Bos GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|54125480 Bos GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|54125508 Bubalus GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|54125464 Syncerus GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi| 119514499 Capra GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGCGGAGGAT 100 gi| 119655282 Ammotragus GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGCGGAGGAT 100 gi|89160951 Ovis GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGCGGAGGAT 100 gi|73697718 Rangifer GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|50442265 Rangifer GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|50442321 Capreolus GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|50442307 Alces GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi| 158714095 Cervus GTGGAGTGACGTCGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|50442285 Cervus GTGGAGTGACGTGGGCCTCTGCAAGAAGCGACCAAAACCTGGAGGAGGAT 100 gi|308194928 Homo ATGGAGTGACCTGGGCCTCTGCAAGAAGCGCCCGAAGCCTG—GAGGAT 91

Fig. 2. Beginning. Alignment of the PRNP gene nucleotide sequences.

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gi|27733849 Equus GGAACACTGGGGGGAGCCGATACCCCGGGCAGGGCAGTCCTGGAGGCAAC 147 gi| 119514511 Equus GGAACACTGGGGGGAGCCGATACCCCGGGCAGGGCAGTCCTGGAGGCAAC 147 gi| 119489983 Sus GGAACACTGGGGGGAGCCGATACCCAGGGCAGGGTAGTCCTGGAGGCAAC 150 gi| 119489801 Bos GGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAAC 150 gi|54125480 Bos GGAACACGGTGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAAC 150 gi|54125508 Bubalus GGAACACTGGGGGGAGCCGATACCCGGGACAGGGCAGTCCTGGAGGCAAC 150 gi|54125464 Syncerus GGAACACTGGGGGGAGCCGATACCCAGGACAGGGCAGTCCTGGAGGCAAC 150 gi| 119514499 Capra GGAACACTGGGGGGAGCCGATACCCGGGACAGGGCAGTCCTGGAGGCAAC 150 gi| 119655282 Ammotragus GGAACACTGGAGGGAGCCGATACCCGGGACAGGGCAGTCCTGGAGGCAAC 150 gi|89160951 Ovis GGAACACTGGGGGGAGCCGATACCCGGGACAGGGCAGTCCTGGAGGCAAC 150 gi|73697718 Rangifer GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi|50442265 Rangifer GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi|50442321 Capreolus GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi|50442307 Alces GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi| 158714095 Cervus GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi|50442285 Cervus GGAACACTGGGGGGAGCCGATACCCGGGACAGGGAAGTCCTGGAGGCAAC 150 gi|308194928 Homo GGAACACTGGGGGCAGCCGATACCCGGGGCAGGGCAGCCCTGGAGGCAAC 141

gi|27733849 Equus CGCTACCCACCCCAGGGCGGTGGCGGCTGGGGTCAACCCCATGGTGGTG- 196 gi| 119514511 Equus CGCTACCCACCCCAGGGCGGTGGCGGCTGGGGTCAACCCCATGGTGGTG- 196 gi| 119489983 Sus CGCTATCCACCCCAGGGAGGGGGTGGCTGGGGACAGCCCCACGGAGGTG- 199 gi| 119489801 Bos CGTTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGG 200 gi|54125480 Bos CGTTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGG 200 gi|54125508 Bubalus CGTTATCCATCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGG 200 gi|54125464 Syncerus CGTTATCCATCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTGG 200 gi| 119514499 Capra CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTG- 199 gi| 119655282 Ammotragus CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTG- 199 gi|89160951 Ovis CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTG- 199 gi|73697718 Rangifer CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGGGGTG- 199 gi|50442265 Rangifer CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGGGGTG- 199 gi|50442321 Capreolus CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTG- 199 gi|50442307 Alces CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCTCATGGAGGTG- 199 gi| 158714095 Cervus CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGTCAGCCCCATGGAGGTG- 199 gi|50442285 Cervus CGCTATCCACCTCAGGGAGGGGGTGGCTGGGGCCAGCCCCATGGAGGTG- 199 gi|308194928 Homo CGCTACCCACCTCAGGGCGGTGGTGGCTGGGGGCAGCCTCATGGTGGTG- 190

gi|27733849 Equus --------GTTGGGGTCAGCCCCATGGTGGTGGCT 223

gi| 119514511 Equus -.....GTTGGGGTCAGCCCCATGGTGGTGGCT 223

gi| 119489983 Sus ........GCTGGGGACAGCCCCACGGAGGCGGCT 226

gi| 119489801 Bos CTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAACCTCATGGAGGTGGCT 250 gi|54125480 Bos CTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAGCCTCATGGAGGTGGCT 250 gi|54125508 Bubalus CTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAACCTCATGGAGGTGGCT 250 gi|54125464 Syncerus CTGGGGCCAGCCTCATGGAGGTGGCTGGGGCCAACCTCATGGAGGTGGCT 250

gi| 119514499 Capra ------GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi| 119655282 Ammotragus .GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|89160951 Ovis ---------GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|73697718 Rangifer .....GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|50442265 Rangifer .....GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|50442321 Capreolus ----GCTGGGGCCAACCTCATGGAGGAGGCT 226

gi|50442307 Alces ........GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi| 158714095 Cervus -----GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|50442285 Cervus -......GCTGGGGCCAACCTCATGGAGGTGGCT 226

gi|308194928 Homo --------GCTGGGGGCAGCCTCATGGTGGTGGCT 217

gi|27733849 Equus GGGGTCAGCCGCATGGTGGTGGTTGGGGACAGCCCCATGGTGGTGGAGGC 273 gi| 119514511 Equus GGGGTCAGCCGCATGGTGGTGGTTGGGGACAGCCCCATGGTGGTGGAGGC 273 gi| 119489983 Sus GGGGACAGCCCCACGGTGGCGGCTGGGGACAGCCCCATGGTGGCGGAGGC 276 gi| 119489801 Bos GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 300 gi|54125480 Bos GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 300 gi|54125508 Bubalus GGGGTCAGCCGCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 300 gi|54125464 Syncerus GGGGTCAGCCCCATGGAGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 300 gi| 119514499 Capra GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi| 119655282 Ammotragus GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi|89160951 Ovis GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi|73697718 Rangifer GGGGTCAGCCCCATGGTGGTGGCTGGGGGCAGCCACATGGTGGTGGAGGC 276 gi|50442265 Rangifer GGGGTCAGCCCCATGGTGGTGGCTGGGGGCAGCCACATGGTGGTGGAGGC 276

Fig. 2. Continued. Alignment of the PRNP gene nucleotide sequences.

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gi|50442321 Capreolus GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi|50442307 Alces GGGGGCAGCCCCATGGTGGTGGCTGGGGGCAGCCACATGGTGGTGGAGGC 276 gi| 158714095 Cervus GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi|50442285 Cervus GGGGTCAGCCCCATGGTGGTGGCTGGGGACAGCCACATGGTGGTGGAGGC 276 gi|308194928 Homo GGGGGCAGCCCCATGGTGGTGGCTGGGGACAGCCTCATGGTGGTG---GC 264

gi|27733849 Equus TGGGGTCAAGGTG—GCTCCCATGGTCAGTGGAACAAGCCCAGTAAGCC 320

gi| 119514511 Equus TGGGGTCAAGGTG—GCTCCCATGGTCAGTGGAACAAGCCCAGTAAGCC 320

gi| 119489983 Sus TGGGGTCAAGGTGGTGGCTCCCACGGTCAGTGGAACAAGCCCAGTAAGCC 326

gi| 119489801 Bos TGGGGTCAAGGTG—GTACCCACGGTCAATGGAACAAACCCAGTAAGCC 347

gi|54125480 Bos TGGGGTCAAGGTG—GTACCCACGGTCAATGGAACAAACCCAGTAAGCC 347 gi|54125508 Bubalus TGGGGTCAAGGTG—GTACCCACGGTCAATGGAACAAGCCCAGTAAGCC 347 gi|54125464 Syncerus TGGGGTCAAGGTG—GTACCCACGGTCAATGGAACAAGCCCAGTAAGCC 347 gi| 119514499 Capra TGGGGTCAAGGTG—GTAGCCACAGTCAGTGGAACAAGCCCAGTAAGCC 323 gi| 119655282 Ammotragus TGGGGTCAAGGTG—GTAGCCACAGTCAGTGGAACAAGCCCAGTAAGCC 323 gi|89160951 Ovis TGGGGTCAAGGTG—GTAGCCACAGTCAGTGGAACAAGCCCAGTAAGCC 323

gi|73697718 Rangifer TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323 gi|50442265 Rangifer TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323 gi|50442321 Capreolus TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323 gi|50442307 Alces TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323

gi| 158714095 Cervus TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323 gi|50442285 Cervus TGGGGTCAAGGTG—GTACCCACAGTCAGTGGAACAAGCCCAGTAAACC 323 gi|308194928 Homo TGGGGTCAAGGAGGTGGCACCCACAGTCAGTGGAACAAGCCGAGTAAGCC 314

gi|27733849 Equus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCGGCAGCTGGGGCAGTGG 370 gi| 119514511 Equus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCGGCAGCTGGGGCAGTGG 370

gi| 119489983 Sus GAAAACCAACATGAAGCATGTGGCAGGCGCCGCTGCAGCTGGGGCAGTGG 376 gi| 119489801 Bos AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 397 gi|54125480 Bos AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 397 gi|54125508 Bubalus AAAAACCAACATGAAGCATATGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 397 gi|54125464 Syncerus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 397 gi| 119514499 Capra AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi| 119655282 Ammotragus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi|89160951 Ovis AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi|73697718 Rangifer AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCCGCTGGAGCAGTGG 373 gi|50442265 Rangifer AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCCGCTGGAGCAGTGG 373 gi|50442321 Capreolus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi|50442307 Alces CAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi| 158714095 Cervus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi|50442285 Cervus AAAAACCAACATGAAGCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGG 373 gi|308194928 Homo AAAAACCAACATGAAGCACATGGCTGGTGCTGCAGCAGCTGGGGCAGTGG 364

gi|27733849 Equus TTGGGGGCCTCGGCGGCTACATGCTGGGGAGTGCCATGAGCAGACCCCTC 420 gi| 119514511 Equus TTGGGGGCCTCGGCGGCTACATGCTGGGGAGTGCCATGAGCAGACCCCTC 420 gi| 119489983 Sus TAGGGGGCCTCGGCGGTTACATGCTGGGGAGTGCCATGAGCAGACCCCTG 426 gi| 119489801 Bos TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 447 gi|54125480 Bos TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 447 gi|54125508 Bubalus TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 447 gi|54125464 Syncerus TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 447 gi| 119514499 Capra TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi| 119655282 Ammotragus TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|89160951 Ovis TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|73697718 Rangifer TAGGGGGCCTCAGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|50442265 Rangifer TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|50442321 Capreolus TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|50442307 Alces TAGGGGGCCTTGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi| 158714095 Cervus TAGGGGGCCTCGGTGGCTACATGCTGGGAAGTGCCATGAGCAGGCCTCTT 423 gi|50442285 Cervus TAGGGGGCCTCGGTGGCTACATGCTGGGAAGTGCCATGAATAGGCCTCTT 423 gi|308194928 Homo TGGGGGGCCTTGGCGGCTACATGCTGGGAAGTGCCATGAGCAGGCCCATC 414

gi|27733849 Equus ATTCATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 470 gi| 119514511 Equus ATTCATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 470 gi| 119489983 Sus ATACACTTTGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGTA 476 gi| 119489801 Bos ATACATTTTGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCA 497

Fig. 2. Continued. Alignment of the PRNP gene nucleotide sequences.

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gi|54125480 Bos ATACATTTTGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCA 497 gi|54125508 Bubalus ATACATTTTGGTAATGACTATGAGGACCGTTACTATCGTGAAAACATGCA 497 gi|54125464 Syncerus ATACATTTTGGTAATGACTATGAGGACCGTTACTATCGTGAAAACATGCA 497 gi| 119514499 Capra ATACATTTTGGCAATGACTATGAGGACCGTTACTATCATGAAAACATGTA 473

gi| 119655282 Ammotragus ATACATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi|89160951 Ovis ATACATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473

gi|73697718 Rangifer ATACATTTTGGCAACGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi|50442265 Rangifer ATACATTTTGGCAACGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi|50442321 Capreolus AT ACATTTTGGCAACGACT ATGAGGACCGTT ACT ATCGTGAAAACATGT A 473 gi|50442307 Alces ATACATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi| 158714095 Cervus ATACATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi|50442285 Cervus ATACATTTTGGCAATGACTATGAGGACCGTTACTATCGTGAAAACATGTA 473 gi|308194928 Homo ATACATTTCGGCAGTGACTATGAGGACCGTTACTATCGTGAAAACATGCA 464

CCGTTACCCCAACCAAGTGTACTACAGGCCGGTAAGTGAGTACAGCAACC 520 CCGTTACCCCAACCAAGTGTACTACAGGCCGGTAAATGAGTACAGCAACC 520 CCGTTACCCCAACCAAGTGTACTACAGGCCAGTGGATCAGTACAGCAACC 526 CCGTTACCCCAACCAAGTGTACTACAGGCCAGTGGATCAGTATAGTAACC 547 CCGTTACCCCAACCAAGTGTACTACAGGCCAGTGGATCAGTATAGTAACC 547 CCGTTACCCCAACCAAGTGTACTACAGGCCAGTGGATCAGTATAGTAACC 547 CCGTTACCCCAACCAAGTATACTACAGGCCAGTGGATCAGTATAGTAACC 547 CCGTTACCCCAACCAAGTGTACTACAGACCAGTGGATCAGTATAGTAACC 523 gi| 119655282 Ammotragus CCGTTACCCCAACCAAGTGTACTACAGACCAGTGGATCAGTATAGTAACC 523 gi|89160951 Ovis CCGTTACCCCAACCAAGTGTACTACAGACCAGTGGATCAGTATAGTAACC 523

gi|73697718 Rangifer CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGT AT AAT AACC 523 gi|50442265 Rangifer CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGT AT AAT AACC 523 gi|50442321 Capreolus CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGT AT AAT AACC 523 gi|50442307 Alces CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGT AT AAT AACC 523

gi| 158714095 Cervus CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGT AT AAT AACC 523 gi|50442285 Cervus CCGTT ACCCCAACCAAGTGT ACT ACAGGCCAGTGGATCAGTAT AAT AACC 523

gi|308194928 Homo CCGTTACCCCAACCAAGTGTACTACAGGCCCATGGATGAGTACAGCAACC 514

gi|27733849 Equus AGAACAACTTTGTGCACGACTGCGTCAACATCACGGTCAAGCAGCACACA 570 gi| 119514511 Equus AGAACAACTTTGTGCACGACTGCGTCAACATCACGGTCAAGCAGCACACG 570 gi| 119489983 Sus AGAACAGTTTTGTGCATGACTGCGTCAACATCACCGTCAAGCAGCACACA 576

gi| 119489801 Bos AGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGGAACACACA 597

gi|54125480 Bos AGAACAATTTTGTGCATGACTGTGTCAACATCACAGTCAAGGAACACACA 597 gi|54125508 Bubalus AGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGGAACACACA 597 gi|54125464 Syncerus AGAACAGCTTTGTGCATGACTGTGTCAACATCACAGTCAAGGAACACACA 597 gi| 119514499 Capra AGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573

gi| 119655282 Ammotragus AGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|89160951 Ovis AGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573

gi|73697718 Rangifer AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|50442265 Rangifer AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|50442321 Capreolus AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|50442307 Alces AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573

gi| 158714095 Cervus AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|50442285 Cervus AGAACACCTTTGTGCATGACTGTGTCAACATCACAGTCAAGCAACACACA 573 gi|308194928 Homo AGAACAACTTTGTGCACGACTGCGTCAATATCACAATCAAGCAGCACACG 564

GTCACCACCACCACCAAGGGGGAGAACTTCACCGAGACCGACGTCAAGAT 620 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAGACCGACGTCAAGAT 620 GTGACCACGACCACCAAGGGGGAGAACTTCACCGAGACGGACGTCAAGAT 626 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 647 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 647 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 647 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACGTCAAGAT 647 GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 623 gi| 119655282 Ammotragus GTCACCACCACCACCAAGGGGGAGAACTTCACTGAAACTGACATCAAGAT 623 gi|89160951 Ovis GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 623 gi|73697718 Rangifer GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATTAAGAT 623 gi|50442265 Rangifer GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATTAAGAT 623 gi|50442321 Capreolus GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATTAAGAT 623 gi|50442307 Alces GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATTAAGAT 623 gi| 158714095 Cervus GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 623 gi|50442285 Cervus GTCACCACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGAT 623 gi|308194928 omo GTCACCACAACCACCAAGGGGGAGAACTTCACCGAGACCGACGTTAAGAT 614

Fig. 2. Continued. Alignment of the PRNP gene nucleotide sequences.

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CATGGAGCGCGTGGTGGAGCAGATGTGCATCACCCAGTACCAGAAAGAGT 670 CATGGAGCGCGTGGTGGAGCAGATGTGCATCACCCAGTACCAGAAAGAGT 670 GATAGAGCGCGTGGTGGAACAGATGTGCATCACCCAGTACCAGAAAGAGT 676 GATGGAGCGAGTGGTGGAGCAAATGTGCATTACCCAGTACCAGAGAGAAT 697 GATGGAGCGAGTGGTGGAGCAAATGTGCATTACCCAGTACCAGAGAGAAT 697 GATGGAGCGAGTGGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 697 GATGGAGCGAGTGGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 697 AATGGAGCGAGTGGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi| 119655282 Ammotragus AATGGAGCGAGTGGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|89160951 Ovis AATGGAGCGAGTGGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|73697718 Rangifer GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|50442265 Rangifer GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|50442321 Capreolus GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|50442307 Alces GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673

gi| 158714095 Cervus GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|50442285 Cervus GATGGAGCGAGTTGTGGAGCAAATGTGCATCACCCAGTACCAGAGAGAAT 673 gi|308194928 Homo GATGGAGCGCGTGGTTGAGCAGATGTGTATCACCCAGTACGAGAGGGAAT 664

ACGAGGCTTTTCAACAAAGAGGGGCGAGCGTGGTCCTCTTCTCCTCCCCG 720 ACGAGGCTTTTCAACAAAGAGGGGCGAGCGTGGTCCTCTTCTCCTCCCCG 720 ACGAGGCGTACGCCCAAAGAGGGGCCAGTGTGATCCTCTTCTCCTCCCCT 726 CCCAGGCTTATTACCAACGAGGGGCAAGTGTGATCCTCTTCTCTTCCCCT 747 CCCAGGCTTATTACCAACGAGGGGCAAGTGTGATCCTCTTCTCTTCCCCT 747 CCCAGGCTTATTACCAACGAGGGGCAAGTGTGATCCTCTTCTCTTCCCCT 747 CCCAGGCTTATTACCAACGAGGGGCAAGTGTGATCCTCTTCTCTTCCCCT 747 CCCAGGCTTATTACCAAAGGGGGGCAAGTGTGATCCTCTTTTCTCCCCCT 723 gi| 119655282 Ammotragus CCCAGGCTTATTACCAAAGGGGGGCAAGTGTGATCCTCTTTTCTTCCCCT 723 gi|89160951 Ovis CCCAGGCTTATTACCAAAGGGGGGCAAGTGTGATCCTCTTTTCTTCCCCT 723

gi|73697718 Rangifer CCCAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi|50442265 Rangifer CCCAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi|50442321 Capreolus CCCAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi|50442307 Alces CCCAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi| 158714095 Cervus CCGAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi|50442285 Cervus CCGAGGCTTATTACCAAAGAGGGGCAAGTGTGATCCTCTTCTCCTCCCCT 723 gi|308194928 Homo CTCAGGCCTATTACCAGAGAGGATCGAGCATGGTCCTCTTCTCCTCTCCA 714

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CCTGTGGTCCTCCTCATCTCTT.............742

CCTGTGGTCCTCCTCATCTCTTTCCTCATTTTCCTCATAGTGGGCTGA 768 CCTGTGATCCTCCTCATCTCTTTCCTCCTTTTCCTCATAGTGGGCTGA 774 CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 795 CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 795 CCTGTGATCCTCCTCATCTCTTTGCTCATTTTTCTCATAGTAGGATAG 795 CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 795 CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi| 119655282 Ammotragus CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|89160951 Ovis CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771

gi|73697718 Rangifer CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|50442265 Rangifer CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|50442321 Capreolus CCTGTGATCCTCCTCATATCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|50442307 Alces CCTGTGATCCTCCTAATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi| 158714095 Cervus CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|50442285 Cervus CCTGTGATCCTCCTCATCTCTTTCCTCATTTTTCTCATAGTAGGATAG 771 gi|308194928 Homo CCTGTGATCCTCCTGATCTCTTTCCTCATCTTCCTGATAGTGGGATGA 762

Notes:

* indicates identical nucleotide sequences;

-, shift of the nucleotide sequences for a more efficient alignment; . or:, nucleotide substitution.

Fig. 2. Ending. Alignment of the PRNP gene nucleotide sequences.

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As follows from Fig. 2, there are point differences at positions 5, 7-12, 16, 21-22, 24, 30, 33, 47, 50, 61, 63, 66, 94, 108, 110, 114, 126, 135, 138, 153, 156, 160, 162, 171, 174, 190, 193, 196, 201-223 (numbered as in Bos taurus protein) between Bos taurus, Bos javancus, Bubalus bubalis, and Syncerus caffer caffer; positions 225, 231, 237, 240, 246, 255, 261, 264, 270, 273, 283, 294, 296-298, 318, 320, 324, 339, 348, 366, 375, 378, 381, 399, 408, 411, 414, 438, 444, 447,453, 456, 459, 462, 496, 528, 532, 540, 543, 554-555, 564, 570, 576, 582, 589, 600, 606, 630, 636, 642, 648, 660, 663, 676,

686, 698-699, 705, 708-709, 721, 723, 726, 738, 741742, 744, 747, 762, 769-795 are different only in

Equus caballus.

To better visualize the level of evolutionary relatedness of the prion protein sequences, a phylogenetic tree presented in Fig. 3 was built in the ClustalW software (see Fig. 2 for designations).

Also, gene sequences of pathogenic and normal prion protein from Ovis aries was performed (Fig. 4). It demonstrated that the nucleotide sequences of PrPc and PrPsc are identical.

Fig. 3. Phylogenetic tree of the PRNP protein gene sequences.

gi|341942290PrPsc

GGGTCAAGGTGGTAGCCACAGTCAGTGGAACAAGCCCAGTAAGCCAAAAACCAACATGAA 60 gi|47028553PrP -

GGTCAAGGTGGTAGCCACAGTCAGTGGAACAAGCCCAGTAAGCCAAAAACCAACATGAA 59 gi|341942290PrPsc

GCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGGTAGGGGGCCTTGGTGGCTACATGCr 120 gi|47028553PrP

GCATGTGGCAGGAGCTGCTGCAGCTGGAGCAGTGGTAGGGGGCCTTGGTGGCTACATGCT 119 gi|341942290PrPsc

GGGAAGTGCCATGAGCAGGCCTCTTATACATTTTGGCAATGACTATGAGGACCGTTACTA 180 gi|47028553PrP

GGGAAGTGCCATGAGCAGGCCTCTTATACATTTTGGCAATGACTATGAGGACCGTTACTA 179 gi|341942290PrPsc

TCGTGAAAACATGTACCGTTACCCCAACCAAGTGTACTACAGACCAGTGGATCAGTATAG 240 gi|47028553PrP

TCGTGAAAACATGTACCGTTACCCCAACCAAGTGTACTACAGACCAGTGGATCAGTATAG 239 gi|341942290PrPsc

AACCAGAACAACTTTGTGCATGACTGTGTCAACACCACAGTCAAGCAACACACAGTCAC 300 gi|47028553PrP

TAACCAGAACAACTTTGTGCATGACTGTGTCAACATCACAGTCAAGCTACACACAGTCAC 299 gi|341942290PrPsc

CACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGATAATGGAGCGAGTGGT 360 gi|47028553PrP

CACCACCACCAAGGGGGAGAACTTCACCGAAACTGACATCAAGATAATGGAGCGAGTGGT 359

gi|341942290PrPsc GGAGCAAATGTGCATCACCCAGTACCAGAGAGAATCCCAGGCTT 404 gi|47028553PrP GGAGCAAATGTGCATCACCCAGTACCAGAGAGAATCCCAGGCT- 402

Fig. 4. Alignment of normal (PrPc) and pathogenic (PrPsc) forms of PRNP prion protein from Ovis aries.

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The phylogenetic analysis confirmed that prion protein sequences are rather conserved and differ only by conformation and relative stability to proteolysis it associates with. This does not allow choosing a DNA target among the prion sequences for further analysis with PCR. Therefore, here we chose a variety of the PCR method, i.e. the real-time immuno-PCR, where DNA molecule is used as a marker, to detect infectious prion proteins. Immuno-PCR allows to detect pathogenic prion protein using specific antibodies labeled with double-strand DNA. Immuno-PCR combines the universality of the enzyme-linked immunosorbent assays with sensitivity of PCR. The

method allows for protein detection at the level of several hundred molecules.

To choose an appropriate antibody reacting with pathogenic prion proteins, we analyzed commercial antibodies. Because of the high inter-species homology noted for the PrP protein, antibodies against peptide conjugates are the most feasible.

Therefore, we chose a mouse monoclonal antibody 15B3 (Prionics) obtained using 3 different sequences (epitopes) of human PrP: 15b3-1 includes amino acid residues 142-148 GSDYEDR(YY); 15b3-2, residues 162-170 YYRPVDQYS; and 15b3-3, residues 214-226 CITQYQRESQAYY (Fig. 5).

gi|56180813Sus

MVKSHIGGWILVLFVAAWSDIGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGG- 59 gi|119514512Equus MVKSHVGGWILVLFVATWSDVGLCKKRPKPGG-

WNTGGSRYPGQGSPGGNRYPPQGGGG- 58 gi|6110615Ovis

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGG- 59 gi|1149617Capra

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGG- 59 gi|34334038Bos

MVKSHIGSWILVLFVAMWSDVGLCKKRPKPGGGWNTGGSRYPGQGSPGGNRYPPQGGGGR 60 gi|89160954Homo --MANLGCWMLVLFVATW SDLGLCKKRPKPGG-

WNTGGSRYPGQGSPGGNRYPPQGGGG- 56

gi|56180813Sus ---

WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGGGSHGQWNKPSKPKTN 112

gi|119514512Equus-WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGG-

SHGQWNKPSKPKTN 110

gi|6110615Ovis ---WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGG-

SHSQWNKPSKPKTN 111

gi| 1149617Capra -WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGG-

SHSQWNKPSKPKTN 111

gi|34334038Bos GQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGGGWGQGG-THGQWNKP SKPKTN 119

gi|89160954Homo --WGQPHGGGWGQPHGGGWGQPHGGGWGQPHGGG-

WGQGGGTHSQWNKPSKPKTN 108

gi|56180813Sus

MKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGSDYEDRYYRENMYRYPNQVYYRPVDQ 172 gi|119514512Equus

MKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVNE 170 gi|6110615Ovis

MKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQ 171 gi|1149617Capra

MKHVAGAAAAGAVVGGLGGYMLGSAMSRPLMHFGNDYEDRYYRENMYRYPNQVYYRPVDQ 171 gi|34334038Bos

MKHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGSDYEDRYYRENMHRYPNQVYYRPVDQ 179 gi|89160954Homo

MKHMAGAAAAGAVVGGLGGYMLGSAMSRPnHFGSDYEDRYYRENMHRYPNQVYYRPMDE 168

gi|56180813Sus

YSNQNSFVHDCVNITVKQHTVTTTTKGENFTETDVKMIERVVEQMCITQYQKEYEAYAQR 232 gi|119514512Equus

YSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDVKIMERVVEQMCITQYQKEYEAFQQR 230

Fig. 5. Beginning. Amino acid sequence of the PRNP prion protein.

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gi|6110615Ovis

YSNQKNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERWEQMaTQYQRESQAYYQR 231 gi|1149617Capra

YSNQNNFVHDCVNITVKQHTVTTTTKGENFTETDIKIMERWEQMCITQYQRESQAYYQR 231 gi|34334038Bos

YSNQNNFVHDCVNITVKEHTVTTTTKGENFTETDIKMMERWEQMCITQYQRESQAYYQR 239 gi|89160954Homo

HSNQNNFVHDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCITQYERESQAYYQR 228

Fig. 5. Ending. Amino acid sequence of the PRNP prion protein.

15B3-1 and 15B3-2 bind beta-sheets that accumulate in PrPsc, and 15B3-3 recognizes amino acid residues near the C-terminus.

15B3 is an antibody specifically recognizing an aberrantly folded PrPsc protein, and not the normal PrP molecules (PrPc).

The Prionics company proved experimentally that 15B3 reacts with pathogenic PrPsc prions of man, cattle, sheep, deer, mouse, and hamster, but does not react with the normal prions. Therefore, 15B3 can be used as a detecting antibody for further analysis.

The procedure goes as follows. Antigen (prion protein) is introduced into a 96-well polysterene plate, 100 gL per well, and the plate is incubated at 37°C for 60 min. To choose the optimal conditions for antigen adsorption on the plastics, the antigen was incubated at concentrations of 0.5, 1, 2.5, 5, 10, 25, and 50 gg/mL for 30, 60, 90, 120, 150, or 180 min. Unbound material is removed from the wells with a simple shaking followed by three washes (washing buffer: 50 mM Tris, 150 mM NaCl, and 0.5 mL/L Tween 20). Nonspecific binding sites are blocked by a 30-min incubation with PBS supplemented with bovine serum albumin, 100 gL per well. After removal of the blocking solution (by washing), biotinylated monoclonal antibody to pathogenic prion protein 15B3 is added, 100 gL per well, to determine the adsorbed material, and the plate is incubated for 2 h at 18°C. Unbound antibodies are removed by a triple washing of the wells with PBS containing 1 mL/L Tween and triple washing with PBS containing 15 g/L bovine serum albumin. To prepare DNA reporter agent, streptavidin-biotin complex was chosen as a binding unit between the antibody and the DNA reporter.

A molecule of streptavidin comprises four identical subunits and is capable of binding four biotin molecules, which allowed using it as a binding unit between two biotin-containing compounds. In this case, DNA tail is also biotinylated, and streptavidin functions as a bridge binding the two molecules containing biotin residues.

Preparation of the conjugates of antibodies and DNA with biotin is accompanied by minimal changes in their immunological activity.

Recombinant streptavidin is pre-incubated for 45 min at 4°C with biotinylated DNA reporter in the molar ratio of 1 : 2. Then, the streptavidin-DNA complex is added to the wells and the plate is incubated for 30 min at room temperature. The wells are washed 5 times with PBS and 10 times, with distilled water, and then subjected to PCR.

By this stage of the study, we have already aligned the PRNP gene nucleotide sequences and have built the phylogenetic tree. Alignment of gene sequences of pathogenic and normal prion proteins from Ovis aries has demonstrated that the nucleotide sequences of PrPc and PrPsc are identical.

To choose a high-performance DNA target, we performed the analysis of GenBank, Sol Genomic Network, and EMBL-EBI databases, which proved that the prion protein gene sequences are rather conserved; therefore, it is not possible to choose a DNA target among the prion sequences for further analysis with PCR. Therefore, we have chosen the real-time immuno-PCR method for detection of infectious prion proteins, where DNA molecule is used as a marker.

Mouse monoclonal antibody 15B3 obtained using three different sequences (epitopes) of the human PrP peptide (15b3 -1 includes amino acid residues 142-148 GSDYEDR(YY); 15b3-2, residues 162-170 YYRPVDQYS and 15b3-3, residues 214-226 CITQYQRESQAYY) was chosen for the work.

It has been shown experimentally that 15B3 reacts with pathogenic PrPsc prions of man, cattle, sheep, deer, mouse, and hamster, but does not react with the normal prions. Therefore, 15B3 can be used as a detecting antibody for further analysis.

The primer design as such is preceded by the construction of a detailed model of the target gene or another nucleotide sequence to be amplified.

To perform the immuno-PCR analysis, a DNA (or a DNA tail) template was needed.

To decrease the risk of false response due to exogenous contamination of DNA in the assay, we designed a DNA tail which does not exist in nature. A synthetic random 194 bp long sequence (fragment length in the range of 150-300 bp is considered optimal) was prepared (see Fig. 6) [11, 12].

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AGGAGGTGGCCACGACTGCGAAGGAGGTGGCGTAGGATAGAGT-

CAGTCCTTGGCCrcCTTGGCCCAGTTAAGAAGTTGCAGCCACA-

CACGCTGTTGTTGGGTTCGGGGCGGAGTTGCAGCCATCTACACAAACGA-

TACCCTCGTGCAGCTGGAGAAGCAGCACGGCCTATTACCTGGAGGAGGATCGAAACTGA

Fig. 6. DNA template sequence.

The created sequence was analyzed in GenBank using the BLAST software to confirm that there are no homologs of the sequence.

One of the key factors in the reaction are the primers, synthetic oligonucleotides 20-30 nucleotide long. Primers are complementary to DNA chains in regions at the boarder of a chosen DNA fragment and are oriented with their 3'-ends facing each other and along the chosen DNA sequence to be amplified. The length of the amplified fragment is determined by the distance between the primers.

In the PCR amplification, two oligonucleotide primers are used. Primers are chosen so that the synthesis by polymerase would proceed only between them, doubling the number of copies of this DNA region. As a result, the amount of a specific fragment grows exponentially.

Primer construction, probably, is the most critical parameter for a successful PCR analysis. Primer sequence determines a whole number of parameters, such as the position and length of the product, its melting temperature, and yield of the product. Poorly

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Table 7. Parameters of the primers

constructed primer may lead to small amount of the product, its absence due to non-specific amplification and/or dimer formation by a primer, which may become a competitive process inhibiting the product formation [11].

Taking into account the above-mentioned issues, the following two 20-nucleotide long primers were selected for the synthesized DNA tail:

>>>>>> left primer - starting from 41 bp-AGTCAGTCCTTGGCCTCCTT;

<<<<<< right primer - starting from 193 bp-CAGTTTCGATCCTCCTCCAG.

Using the Primer3 software, melting temperature (tm) and other parameters of the primers were chosen (Table 7).

The melting temperature of the left primer tm = 59.8°С and the right primer tm = 60.25°С.

Annealing temperature is set 4-5°С below the melting temperature.

Therefore, the optimal annealing temperature in the amplification program will be ta = 56°С for the left primer and ta = 55.8°С, for the right one.

Primer type Starting position, bp CG, % Length, bp t °С m Sequence

Left primer 41 55 20 60.25 AGTCAGTCCTTGGCCTCCTT

Right primer 193 55 20 59.80 CAGTTTCGATCCTCCTCCAG

Table 8. Final characteristics for primer construction

Main requirements to the primers Values Comparison of the characteristics of chosen primers with the requirements

Primer length from 15 to 30 nucleotides 20 bp, fits

GC content from 45% to 55% 55%, fits

Melting temperature (tm) from +55°C to +75°C tm = 60.25°C and tm =59.80°C fits

Annealing temperature (ta) 4-5 degrees below the melting temperature ta =56°C и ta =55.80°C, fits

Secondary structure of the primer Primer should not fold into a secondary structure with melting temperature equal to or above the tm of the primer Fits (verified using the Mfold 3.2 software package)

Secondary structure of the target site Target site should not fold into a secondary structure with melting temperature equal to or above the tm of the primer Fits (verified using the Mfold 3.2 software package)

Homo- and heterodimerization of the primers Excluded, especially at the 3'-end Fits (verified using the Hybrid software package)

Primer specificity The degree of complementarity to the target site is close to 100%; less than 70% homology with other nucleotide sequences Fits (verified using the BLAST software)

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Therefore, at this stage of the study, random synthetic target DNA sequence (DNA tail) 194 bp long has been created. Analysis of the GenBank using the BLAST software demonstrated that the created sequence has no homologs among the sequences of the database.

Two 20-bp primers were synthesized for the DNA tail. Using the Primer3 software, primer parameters were chosen.

Studies of the specificity of the developed PCR system were performed by the example of meat chop containing the mixture of muscle tissues of beef and pork and supplemented with 1.0, 2.0, 5.0, 10.0, and 15.0 pork meat infected with a pathogenic prion protein. Each stage of DNA isolation was accompanied by the addition of an internal standard. The presence of the pathogenic prion protein in pork tissues was confirmed using the commercial TeSeETM ELISA test-system.

The analysis demonstrated high specificity of the developed PCR system: no non-specific response to 100-% fish flour or chicken chop, as well as their mixtures, was registered. The results are presented in

Fig. 7.

M 12345 678m

M 12345 678 M

Fig. 7. Evaluation of the specificity and sensitivity of the test-system: М, marker; 1, 1.0% infected pork meat in the meat chop; 2, 2.0% infected pork meat in the meat chop; 3, 5.0% infected pork meat in the meat chop; 4, 10.0% infected pork meat in the meat chop;

5, 15.0% infected pork meat in the meat chop; 6, 100% meat chop; 7, fish flour; 8, chicken chop.

Besides, the specificity of the developed test-system was studied based on the comparative analysis of the results on determination of the pathogenic prion protein obtained using the proposed PCR test-system and a commercially available ELISA assay TeSeETM. Over 200 samples of clinical material were tested in parallel. Test results of the commercially available TeSeETM assay and the proposed test-system matched in 198 out of 200 cases. Sensitivity of the reference (commercially available) method was 96.5%. In five of the positive samples not detected by the TeSeETM ELISA assay, initial DNA target concentration did not exceed 100 copies/mL. Therefore, the higher stability of detection of low DNA concentrations in the PCR method, if compared with other methods, is confirmed by the results of clinical samples study (Table 9).

Table 9. Comparison of the results of pork testing using the PCR test-system and the TeSeETM ELISA assay

Number of analyzed samples

PCR test-system TeSeETM ELISA assay

«+» «-» «inh» «+» «-» «inh»

«+» n = 60 «-» n = 140 58 1 1 57 2 1

1 139 0 2 134 4

Relative specificity 96,5 96,5

Notes: «+», positive samples; «—», negative samples; «inh», inhibited samples.

Comparison of the results obtained with the proposed PCR test-system and the reference method evidence real-time high specificity of the developed PCR method.

Therefore, high specificity of the developed test-system and oligonucleotide primers was confirmed by three ways: 1) using the Primer3 software; 2) by electrophoretic separation of the meat chop samples with different percent content of pork tissues infected with a pathogenic prion protein; and 3) by comparative analysis of the results of pathogenic prion protein determination using the proposed PCR test-system and a commercial ELISA assay TeSeETM.

REFERENCES

1. Wickner, R.B., Edskes, H.K., Roberts, B.T., Baxa, U., Pierce, M.M., Ross, E.D., and Brachmann, A., Prions: Proteins as Genes and Infectious Entities, Genes Dev., 2004. V. 18, no. 5, P. 470-485.

2. Heikenwalder, M., Julius, C., Aguzzi, A., Prions and Peripheral Nerves: A Deadly Rendezvous, J. Neurosci., 2007. V. 85, P. 2714-2725.

3. Weissmann, C., and Aguzzi, A., Approaches to Therapy of Prion Diseases, Annu. Rev. Med., 2005. V. 56, P. 321-344.

4. Bartz, J.C., Dejoia, C., Tucker, T., Kincaid, A.E., and Bessen, R.A., Extraneural Prion Neuroinvasion without Lymphoreticular System Infection, J. Virol., 2005. V. 79, P. 11858-11863.

5. Vana, K., Zuber, C., Nikles, D., and Weiss, S., Novel Aspects of Prions, Their Receptor Molecules, and Innovative Approaches for TSE Therapy, Cell. Mol. Neurobiol., 2007. V. 27, no. 1, P. 107-128.

6. Elementnyi ekspress analizator Rapid N cube. Kratkoe rukovodstvo (Express elemental analyzer Rapid N-cube. Brief manual) (Moscow, 2009).

7. Prosekov, A.Yu., Babich, O.O., and Sukhikh, S.A., Sovremennye metody issledovaniya syr’ya i biotekhnologicheskoi produktsyi: Laboratornyi praktikum (Modern methods of investigation of raw materials and biotechnology products: Laboratory course), (Kemerovo Institute of Food Science and Technology, Kemerovo, 2012).

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8. http://www.ncbi.nlm.nih.gov/genbank/

9. Prusiner, S.B., Prions, Proc. Natl. Acad. Sci. USA, 1998. V. 95, no. 23, P. 13363-13383.

10. Apetri, A.C., and Surewicz, A.C., Kinetic Intermediate in the Folding of Human Prion Protein, J. Biol. Chem., 2002. V. 277, P. 44589-44592.

11. Patrushev, L.I., Iskusstvennye geneticheskie sistemy. T. 1. Gennaya i belkovaya inzheneriya (Artificial genetic systems. V. 1. Gene and protein engineering) (Nauka, Moscow, 2004).

12. Aguzzi, A., Sigurdson, C., Heikenwalder, M., Molecular Mechanisms of Prion Pathogenesis, Annu. Rev. Pathol. Mech. Dis., 2008. V. 3, P. 11-40.

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Theory and practice of prion protein analysis in food products Текст научной статьи по специальности «Биологические науки»

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