荷斯坦奶牛杀菌/通透性增加蛋白氮端基因原核表达载体的构建

Construction of Procaryotic Expression Vector of Bactericidal/permeability-increasing Protein N-terminal Gene of Holstein Cow

根据高恒[5]的方法,应用RT-PCR技术,从荷斯坦奶牛嗜中性粒细胞mRNA中扩增出杀菌/通透性增加蛋白N端DNA,将该序列与原核表达载体pGEX-4T-1连接,构建重组质粒pGEX-4T-1-BPI。重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达。实验结果表明获得了BPIN端长度为714bp的DNA序列,序列分析证实在第503位出现了点突变。重组质粒诱导表达后,经SDS-PAGE电泳检测重组蛋白约在52kD处出现特异性蛋白条带,与目的蛋白的分子量一致。表明实验成功地获得了BPI重组蛋白。

According to the method provided by Gao Heng,the N-terminal gene was amplified by RT-PCR from mRNA which were extracted from the polymorphonuclear neutrophils of holstein cow. The purified DNA was connected with expression vector pGEX-4T-1, constructed recombinant plasmid pGEX-4T-1-BPI, which was transformed into E.Coli BL21. It could produce fusion protein induced with IPTG. The results showed that A 714 bp fragment was acquired, and there was a basic mutated group in 503rd compared with the reports. The E.coli with recombinant plasmid was induced with IPTG. The products were analyzed by SDS-PAGE, and a new protein of 52kD was detected. Its molecular weight was the same as expected. In conclusion, the purified recombinant BPI was obtained successfully in our experiment.