鸡Prnp基因原核表达载体的构建及其在大肠埃希菌中的表达

试验旨在构建鸡Prnp基因原核表达载体,并在大肠埃希菌中进行表达,为制备鸡朊蛋白单克隆抗体提供材料。根据GenBank已报道的鸡Prnp基因组序列和pET-28a质粒多克隆位点设计引物,以健康的鸡全血基因组DNA为材料,采用PCR的方法扩增鸡的Prnp基因,将目的基因片段与pET-28a载体连接,构建重组原核表达载体。重组菌转化到E. coli BL21(DE3)感受态细胞中,并用异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactoside,IPTG)进行诱导表达,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达的重组蛋白。结果表明,重组蛋白在诱导剂的终浓度为0.08 mmol·L^-1,16 ℃,220 r·min^-1诱导7 h,蛋白表达量最高。综上所述,本研究成功构建了pET-28a-ChPrnp重组表达菌株,为Prnp朊蛋白的结构、生理功能和致病机制研究提供方法。

Construction of prokaryotic expression vector of chicken Prnp gene and expression in Escherichia coli

In order to construct the prokaryotic expression vector of chicken Prnp gene and express it in Escherichia coli, and to provide materials for the preparation of monoclonal antibodies against chicken prion protein, primers were designed according to chicken Prnp genome sequence reported by GenBank and the polyclonal site of pET-28a plasmid. Using healthy chicken whole blood genomic DNA as material, the Prnp gene of chicken was amplified by PCR. The target gene fragment was connected with pET-28a vector to construct recombinant prokaryotic expression vector. The recombinant bacteria were transformed into E. coli BL21(DE3) competent cells and induced by isopropyl-β-D-thiogalactoside (IPTG). The expressed recombinant protein was identified by SDS-PAGE. The results showed that the expression of recombinant protein was the highest at the final inducer concentration of 0.08 mmol·L ^-1, 16 ℃, 220 r·min^-1 for 7 hours. To sum up, the recombinant expression strain of pET-28a-ChPrnp was successfully constructed in this study, which provides a method for the study of the structure, physiological function and pathogenic mechanism of Prnp prion protein.