Cloning And Prokaryotic Expression Of Sheep And Goat Prion Protein Genes

The total DNAs were extracted from peripheral whole-blood of three Tibetan sheep and three goats, respectively. The PrPc genes were amplified by polymerase chain reaction, using two pairs of primers, respectively, and then cloned into pMD18-T Vector. Sequencing showed that the genes were 771 bp in length. The entire PrPc coding sequences which contains are the complete open reading frames (ORFs), and are a single exons. The genes share very high homology with the published gene sequences. The sequences of the sheep and goat PrP genes, which encode the octapeptide or the onoapeptide, contain five copies of the short G-C-rich element. Comparison of the genes each other revealed that the nucleotide and amino acid identities between them ranged from 99.0% to 99.9% and from 97.7% to 99.6%, respectively. Of the total sixteen bases substitutions, seven bases substitutions of the PrPc genes were synonymous mutation and nine substitutions were amino acid mutation. Except for S240P of SY01,SY02 and SY03, all other amino acid mutations were MYOl's Ml 121 and G129S, MY02's T191R, MY03's G127S, SY02's H143R and R211G, and lied in residues 91 -233 of the C-terminal structural domain. It can be inferred that S240P has breed specificity in goats.To construct two recombinant plasmids, the DNA fragements of the sheep(Ov) PrP gene were amplified secondly by polymerase chain reaction. After being digested with EcoR I and Xho I, they were inserted to the vectors pGEX-4T-l orientally, and then transformed into E.coli BL21(DE3) cells. The monoclone were selected and identified by means of restriction analysis, PCR and sequencing. As the result, the two recombinant plasmids with the different DNA fragements of the PrP gene were constructed and designated as pMY01[Ov rPrP(23-244)] and pMY02[Ov rPrP(91 -244)], respectively. The bacteria containing the recombinant plasmids were induced with IPTG and samples of bacteria culture fluid were collected at different time respectively. After being treated properly, the samples were examined by SDS-PAGE and Western blotting.The results showed that the interest genes were successfully expressed in E.coli and 50.4 ku and 42.9 ku recombinant proteins,which can be recognized by 4C11 and were sensitive to proteinase K treatments, were obtained. It was conformed by gel thin layer scanning analysis that the amount of target proteins occupied 30%-45% of the total proteins.