犬瘟热病毒N基因原核表达质粒的构建

摘要

为了对N蛋白表达作为诊断用抗原、检测犬瘟热病毒(CDV)特异性抗体提供参考依据,以已构建的含犬瘟热病毒N基因的pMD18-N质粒为模板,利用特异性引物进行PCR扩增,得到1572 by的N基因开放阅读框,将所得N基因克隆至经相同双酶切处理后的pET32a(+)原核表达载体中,获得重组质粒pET32a(+)-N,并转化至大肠杆菌BL21(DE3)中进行诱导表达.测序结果显示,目的基因插入位置和阅读框正确,经IPTG诱导表达出分子量约为75 kDa的重组N蛋白.%To offer accordance with N protein expression as diagnosis antigen and detecting CDV specific antibody, based on the constructed pMD18-N plasmid containing canine distemper virus N gene, the open reading frame of 1572bp was amplified by polymerase chain reaction (PCR) using one pair of primers. N gene and pET32a(+)were digested with the same procedure, then N gene was cloned into pET32a(+). The positive recombinant plasmid pET32a(+)-N was identified and transformed into E. coli BL21(DE3). The sequencing results showed that N gene was in the correct inserted position and open reading frame,and a fusion protein about 75 kDa was expressed with IPTG.