黑胸大蠊浓核病毒磷脂酶A2功能区的克隆及其表达.pdfVIP

21 2 21 2181-185 2006 3 VIROLOGICA SINICA March 2006 A2 * ** 430072 Cloning and Expression of Phospholipase A Domain of Periplaneta 2 fuliginosa Densovirus in E.coli YU Hai-yangZHANG Jia-minYANG BoJIANG HongZHOU Liang,LU Jie CHENG Wu-guoLI Dou-linHU Yuan-yang** State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, 430072, China Abstract: 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, SDSindicated 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. Key words: Periplaneta fuliginosa densovirus PfDNVphospholipase A2 Prokaryotic expression RT-PCR PfDNV VP1 A PL A pMD18-T 2 2 pET28a pET26b pET28a-PLA pET26b-PLABL21-codonplusDE3-RILIPTG SDS Ni-NTA western blot PfDNV PLA2 PfDNVA2 Q786A1003-5125(2006)02-0181-05