伪狂犬病病毒闽A株糖蛋白gD基因去信号肽片段在大肠埃希氏菌中的表达

Expression of gD gene encoding area of pseudorabies virus Fa strain exclude signal peptide in Escherichia coli

应用PCR方法从包含伪狂犬病病毒(PRV)Fa株糖蛋白gD基因的重组质粒pB13中扩增出糖蛋白gD基因去信号肽片段,将其克隆到大肠埃希氏菌表达载体pThiohisA中。测序结果表明,糖蛋白gD基因去信号肽片段长1 155 bp,与PRV Fa株糖蛋白gD基因完全一致。经1mmol/L IPTG诱导后,重组质粒pThiohisA-gD在大肠埃希氏菌XL1-blue、Top10、DE3和DH5α中均得到表达,其中在宿主菌XL1-blue中表达量最高。Western-blotting结果显示,大肠埃希氏菌XL1-blue表达的糖蛋白gD具有免疫原性。

A pair of primers was designed and synthesized according to glycoprotein D gene sequence of pseudorabies virus Fa strain, The gD gene encoding area of pseudorabies virus Fa strain exclude signal peptide was amplified by PCR from the recombinant plasmid pB13, which harbors the complete gD gene. The PCR product was cloned into an expression vector pThiohisA for E. coli and then sequenced. The sequencing results showed that gD gene encoding area exclude signal peptide is composed of 1155 bp, which is completely same with the sequence of gD gene of PRV Fa strain. The recombinant plasmid pThiohisAgD was transformed into XL1 blue, Top10,DE3 and DH5a respectively, then the expression was induced by adding IPTG to a final concentration of 1 mmol/L. SDS-PAGE result indicated the expression level was too low, and the expression level in XL1-blue was the highest. Western-blotting further confirmed glycoprotein D expressed in E. coli was antigenic.