Preparation Of Prion Polyclonal Antibodies And Cloning And Analysis Of Prnp Gene Of Two Animals

The definite diagnosis for human and animal prion diseases depends on the detection of PrPSc and/or examination of special pathological changes in the brain tissues of suspected cases.Obtaining of PrP antibody with good specificity and sensitivity is essential for the diagnosis of prion diseases.In this study,we prepared a PrP specific polyclonal antibody(pAb P54)in a specie of Prnp knockout mice via immunization of recombinant human PrP protein residue 23-231,the ELISA reactive titer of pAb P54 was estimated as 1:20480.Then we further verified the specifity of the prepared antibody in Western blot,immunohistochemistry and immunofluorescent assays.Western blots illustrated that the newly prepared pAb P54 could react well with recombinant PrP protein,normal brain PrPC from healthy rodents and human.At the same time,the pathological PrPSc in the brains of various scrapie infected experimental rodents,including 139A-,ME7-and S15-infected mice and 263K-infected hamsters and the different types of human prion diseases,including sCJD,G114V gCJD and FFI also can be well reacted.The electrophoretic patterns of brain PrPC and PrPSc in the reactions with pAb P54 were almost same as those produced by commercial PrP monoclonal antibodies(6D11 and 3F4).Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions with pAb P54.Immunohistochemical(IHC)assays showed that the prepared antibody can specifically recognize abnormal PrPSc deposits in brain slices of 139A-infected mice and 263K-infected hamsters.Immunofluorescent tests with pAb P54 also showed clear green signals surrounding the blue stained cell nuclei.Above results showed that the prepared pAb P54,with reliable specificity and sensitivity,displays an good application prospect not only in the studies of prion protein biology,but also in the detection of brain PrPSc from human beings and rodents.Prnp gene encodes PrP protein,which is conservative among different mammalians and its amino acid sequence associates with the susceptibility of prion disease.Vulpes corsac belongs to canine family,fox genus,which mainly inhabits steppe,desert and semi-desert areas,widely distributes in Central Asia and Western China.They usually taking rodents and birds as the main source of food,meanwhile,many other carnivores may prey them.Ochotona curzonia belongs to ochotonidae family,ochotona genus which is the endemic and key species in Qinghai-Tibet Plateau,can only be seen on alpine meadows and glasslands.They are the main prey of most small and medium-sized carnivores and almost all raptors on the grasslands In this report,we have for the first time cloned and sequenced the Prnp sequence of Vulpes corsac and Ochotona curzonia in Qing-Tobent plateau.Based on the sequencing results,the corresponding amino acid sequences were derived by software and the results of sequence analysis and alignment were also shown.The results showed that the coding sequence of the tested Vulpes corsac's Prnp gene was 774 bp long,which may encode 257 amino acids.The Prnp sequence of the tested Ochotona curzonia was 759bp long and may encode 252 amino acids.Moreover,on the basis of obtaining the correct Prnp sequences of these two species,a deep comparation of the relationship between them and other animal species was carried out.Compared with the published data of foxes,including red fox,Swift fox and Arctic fox,the amino acid sequence of Vulpes corsac PrP was 100%homology.The amino acid sequence of Ochotona curzonia and American pika differ only in one amino acid position.The taxa relationship of these two animals PrP with other species of animals,including human,canine,bovine,cervus,capra,ovis,camelus,felis,Mustela,mouse and hamster were also analyzed.The sequence similarities between these two animals and other animal species,including humans,dogs,cows,deer,sheep,camels,cats,mice,and hamsters were also analyzed.Here,we first obtain the sequence of Vulpes corsac and Ochotona cuezonia and have been submitted to the NCBI for international reference in relevant research fields.As a middle link of food cycle in a special geography,sequencing and understating the sequences of Vulpes corsac and Ochotona cuezonia prion proteins may help for evaluation their potentials in the circulation of prions in a special region.