PRNP Allele Polymorphisms At Codons 136, 154 And 171 In Tan-Han Crossbreed Sheep

【Objective】In order to establish the PCR-SSCP analysis method which used to PRNP allele polymorphism analysis, PCR-SSCP was to be optimized by using amplificated products of PRNP allele as research object. PRNP allele polymorphisms of 104 Tan-Han crossbreed sheep of Yongchang sheep farme in Gansu province were to be analysised by PCR-SSCP method so as to understand PRNP allele frequency of codons 136, 154 and 171 and to estimate the possibility of sheep infected by scrapie. Meantime, sheep with ARR homozygote or heterozygote were to be screened so as to provide for anti-scrapie breeding plan.【Method】The DNA had been extracted using DNA extraction kit and fragments of PRNP allele were amplificated by special primers. (1) Factors such as gel concentration, crosslinking degree, glycerol concentration, voltage, electrophoresis time, proportion of PCR product and loading dye, ionic strength of running buffer, PCR product loading amount had been optimizated by using PCR products to establish PCR-SSCP analysis method. (2) The PCR products had been detected by agarose gel electrophoresis and purified and cloned into pMD-18T and transformated into E. coli DH5α, then, the transfored mixture were cultivated on LB medium (Amp+) and white transparent colonies were selected. Positive clones had been detected by PCR, enzymatic digestion and SSCP and were sequenced. Results of sequencing were analized by DNA Star and the PRNP genotype at codons 136, 154 and 171 were established. PRNP genotype frequency was analized by statistics and scrapie susceptibility of this flock was estimated.【Results】The optimization test of PCR-SSCP showed that the optimal condition was 12% gel concentration, 49:1 crosslinking degree, 0% glycerol concentration, 200 voltage, PCR product/loading dye (1:4), 0.5×TBE, PCR product loading amount≥2.0μL and <4.0μL, and electrophoresis for 45 hours in 4℃. Fifteen genotypes of PRNP allele at codons 136, 154 and 171 were detected in the analyzed animals. In results, high scrapie susceptibility genotypes (ARQ/ARQ, n=38; ARQ/ARH, n=11; VRQ/ARQ, n=5; ARH/ARH, n=2) were 53.85% (56/104), semi-resistant genotypes (ARR/ARQ, n=12; ARR/ARH, n=1) were 12.50% (13/104), resistant genotype (23 ARR/ARR) was 22.12% (23/104) and other genotypes (ARK/ARQ, n=4; ARK/ARH, n=2; AHR/ARQ, n=1; TRQ/TRQ n=1; TRQ/ARR, n=1; TRQ/ARQ, n=1; ARQ/ARP, n=1; ARK/ARK, n=1) of unknown relationship with scrapie were 11.54% (12/104). ARQ/ARP genotype was firstly found. Additionally, there were 18 mutations at other codons in which L141F, R167G, N176Y and Q189L were reported before and Y131N, L133P, G134R, E149V, M157T, Y158H, N162P, Y166D, F178S/L, C182S,E199V/M/L, N200I, F201S and M209T were new mutations found in this research and E189L (33/208), R167G (9/208) and L141F (8/208) were the mutations most frequently observed, the others had a low frequency (about 0.48～1.92%).【Conclusion】The PCR-SSCP method established in this research is confirmed to be stationary and reliable in the PRNP allele polymorphisms analysis. High scrapie susceptibility genotypes of PRNP at codons 136, 154 and 171 in 104 healthy Tan-Han crossbreed sheep were observed in this breed with predominance of the ARQ/ARQ, ARQ/ARH, VRQ/ARQ and ARH/ARH alleles (53.85%,56/104), and this flock blong to the population with high scrapie susceptibility/outbreaks. Therefore, there would be a high risk of the scrapie if prion disease spreads.