家蚕核型多角体病毒ORF 75基因的克隆及表达

根据GenBank发表的序列(L33180)设计引物,利用PCR技术扩增了编码家蚕核型多角体病毒(Bombyxmori Nucleopolyhedrovirus,BmNPV)ORF75基因的DNA片段,将其连接至pMD18-T载体上,获得了该基因成熟蛋白阅读框序列.利用设计的酶切位点将目的基因从克隆载体上切下.表达质粒pGEX-4T-2经BamHI/XhoI双酶切,与目的片断连接,得到重组表达质粒pGEX/Bm75.将重组表达质粒pGEX/Bm75转化入大肠杆菌BL 21中,经IPTG诱导表达后,12%SDS-PAGE分析表明产生57 kDa左右的特异蛋白质条带.对表达产物进行Westernblotting分析,结果表明,pGEX/Bm75经IPTG诱导产生的57 kDa蛋白条带与抗体发生了很强的交叉反应,融合蛋白得到了表达.

Cloning and Expressing of ORF 75 gene of Bombyx mori nucleopolyhedrovirus

based on primers designed according to sequence(L 33180) published on GenBank and Bombyx mori nucleopolyhedrovirus(BmNPV),DNA fragment of BmNPV ORF 75 gene was obtained using PCR amplification and was cloned into pMD18-T vector.The mature protein open reading frame of the DNA was obtained.The fragment was digested by two restriction enzyme BamHI/XhoI,and was joined with pGEX-4T-2 plasmid which was digested by BamHI/XhoI.The pGEX/Bm 75 was obtained,and pGEX/Bm 75 was transformed into BL 21.By inducing with IPTG,the GST-Bm 75 fusion protein was checked by 12% SDS-PAGE(SDS polyacrylamide gel electrophoresis) and Western bloting.The result showed that the molecular weight of fusion protein was about 57 kDa with a GST tag,and the fusion protein was expressed which provide a convenient way for seperation and purification of this protein.