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.
prion, protein, encephalopathy, safety, quality, PCR, analysis
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 non-infectious vitally important protein present in the organism of mammals, including human, and an infec-tious protein, which is a mutation of the normal prion protein causing prion diseases of animals and man.
Prion diseases are a group of transmissive neuro-degenerative 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 µL of bulk volume of a sample to an Eppendorf tube (5–7 mm from the tube bottom). To prevent cross-contamination, the grinding instruments were used once, washed carefully, and sterilized.
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).
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.