黑胸大蠊浓核病毒磷脂酶A2功能区的克隆及其表达

通过RT-PCR扩增获得PfDNV结构蛋白基因VP1含磷脂酶A2(PLA2)功能区片段,将其连接到pMD18-T载体上并亚克隆到原核表达载体pET28a和pET26b,构建阅读框架正确的重组表达载体pET28a-PLA和pET26b-PLA,转化大肠杆菌BL21-codonplus(DE3)-RIL,经IPTG诱导,SDS-PAGE显示得到了目的融合蛋白,以抗组氨酸的单克隆抗体对经Ni-NTA亲和层析柱纯化的目的蛋白进行了westernblot鉴定,结果表明成功表达PfDNV结构蛋白PLA2,对于研究该酶的生物学特性及其在病毒对细胞侵染过程中的功能奠定了基础。

Cloning and Expression of Phospholipase A2 Domain of Periplaneta fuliginosa Densovirus in E.coli

The phospholipase A2 functional domain of PfDNV capsid gene VP1 was obtained by RT-PCR amplification. The amplified fragment was ligated into a pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The recombinant plasmid pET28a-PLA and pET26b-PLA were used to transform E. coli BLl21-codonplus(DE3)-RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion protein was produced. The fusion protein was purified with Ni-NTA affinity columns and analyzed by Western blot using mouse anti-His monoclonal antibodies. The results demonstrated that the recombinant PLA2 protein of PfDNV capsid gene was successfully expressed, which should facilitate further studies on the biological properties of the enzyme and its function in virus infection.