Cloning And Expression Of PK Resistant Core Of PrP From Little-Fat-Tail Sheep In E.coli

The core segment of PrP gene named as PrP435 was amplified from the ovine genomic DNA from the ovine whole blood with the polymerase chain reaction. PrP435 and pET-32a (+) plasmid DNA was digested with restriction enzyme EcoR I and Hind III, purify with SpinPrep Gel DNA Kit as the instruction of the kit and ligate PrP435 and pET~32a (+) to construct a recombinant plasmid which was named as PrP-pET-32a(+). Then the PrP-pET-32a(+) was transformed into E. coli DH5 a . The positive clones was identified and seleted by colony PCR and the open reading frame(ORF)of PrP43swas verified by sequencing. The result showed that it had correct ORF.PrP-pET-32a(+) was transformed into host E.coli BL21(DE3) which carrying T7 RNA polymerase gene (DE3 lysogen). The stability of the plasmid was tested by different temperature, ampicillin concentration and media. The results indicated that the recombinant plasmid was stabile in E.coli BL21(DE3) when it was cultured in TB media containing 200 ug/ml ampicillin concentration at 25℃, and the Trx-PrPc27-30 fuse protein could be expressed.Target protein (Trx-PrPc27-30) could be induced at optimize condition as following : E. coli BL21 (DE3) were incubated in TB containing 200 ug/ml ampicillin at 25℃, until the OD600 value of the bacteria was 1.5, IPTG was added at 1 mM final concentration , and the bacteria were incubated for 4h, the yield of Trx-PrPc27-30 reached the highest. It was about 49. 6% of the totle bacteria protein based on normalized SDS-PAGE analysis. Final,BugBusterTM Protein Extraction Reagent kit and sonication were used to anysis the soluble cytoplasmic fraction and insoluble cytoplasraic fraction. The results indicated almost whole Trx-PrPc27-30 were expressed at insoluble cytoplasmic. The Trx-PrP?7-30 was purified by Ni-NTA His. Bind Resin and identified by Western-blot with monoclonal antibody (F89).