【摘要】 参照GenBank中安哥斯牛杀菌/通透性增加蛋白(BPI)序列,设计合成1对引物。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,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(Escherichia coli)BL21(DE3),用IPTG诱导表达。结果表明,获得了BPIN端长度为714bp的cDNA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。更多还原

【Abstract】 According to the Bactericidal/permeability-increasing Protein(BPI) sequence of Angus calf in GenBank,a pair of primers were designed.The N-terminal cDNA was amplified by RT-PCR from mRNA which was extracted from the polymorphonuclear neutrophils of Hostein cow.The purified cDNA was connective with expression vector pGEX-4T-1 to construct recombinant plasmid pGEX-4T-1-BPI,which was transformed into Escherichia coli BL21.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 52 kD was detected.Its molecular weight was the same as expected,showing the purified recombinant BPI was obtained successfully.更多还原