【作者】 俞海洋；

【导师】 胡远扬；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 硕士

【摘要】 黑胸大蠊是在我国城乡室内分布的主要蟑螂种类,黑胸大蠊浓核病毒(Periplaneta fuliginosa Densovirus, PfDNV)是国内外第一个分类鉴定的蟑螂浓核病毒。其病毒理化性质、基因组结构分析,与组织病理学方面的研究均已完成。但长期以来,由于缺乏稳定的PfDNV体外感染的细胞模型,PfDNV结构蛋白的功能及其复制侵染的分子机理仍不清楚。 研究发现,细小病毒的结构蛋白中具有磷脂酶A2(PLA2)基序与分泌型磷脂酶(如:蛇毒和蜂毒等)的基序有较高的同源性,包含保守的钙离子结合位点YXGXG和催化活性位点HDXXY。插入缺失突变表明,该功能域在病毒粒子的侵染过程中发挥调控作用。 据此,为了进一步研究黑胸大蠊浓核病毒中磷脂酶A2功能域的功能,我们通过聚合酶链式反应(PCR)扩增获得PfDNV含磷脂酶A2(PLA2)功能域片断,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,pET28a-PLA在一般条件下诱导获得包涵体,pET26b-PLA通过优化表达条件在细胞质和周质空间里获得了重组蛋白的可溶性表达形式,以Ni-NTA亲和层析柱在非变性条件下纯化了目的蛋白,以卵磷脂为底物测定其磷脂酶A2活性,对结果进行初步比较分析,结果成功表达PfDNV重组蛋白PLA2,这些初始工作为下一步抗血清的制备研究PfDNV磷脂酶A2功能域在病毒粒子中的存在形式和通过定点突变等方法对其生物学特性的深入研究奠定了基础。更多还原

【Abstract】 Periplaneta fuliginosa is the major species of cockroach distributed in urban and rural area of our country. Periplaneta fuliginosa Densovirus（PfDNV） is the first classified and authenticated cockroach densovirus. Although the research on the biophysical properties, genomic structural analysis and Cytopathology of the virus have been accomplished, the function of structural protein and the molecular mechanism of its infection are still unknown in case of the absence of the stable cell model of PfDNV infection in vitro.Recently it was reported that a phospholipase A2 motif was discovered in the structural protein sequences of parvovirus. The parvovirus phospholipase A2 motif shared amino acid homology with the secreted phospholipase A2 motif（eg, snake venom and bee venom）, including the conserved calcium binding loop YXGXG and the catalytic helix site HDXXY. Also, The insertion and deletion experiment has showed that the function of this PLA2 domain was related to infection of virions.So, in order to get further information ,we study the PfDNV phospholipase A₂ functional domain in protein level. The fragment was obtained by PCR amplification. The amplified products was ligated with pMD18-T vector and sub-cloned into prokaryotic expression vector pET28a and pET26b. The in-frame sequence confirmed recombinant plasmid pET28a-PLA and pET26b-PLA were transformed into E. coli BL21-codonplus-（DE3） -RIL competent cells. After induction by IPTG, SDS-PAGE indicated that the highly expressed fusion proteins were produced. By optimazing the expression condition, we get soluable fusion protein in cytoplasm and periplasm through the induction of pET26b-PLA plasmid. The target fusion protein was purified with Ni-NTA affinity column, then analyzed the PLA2 enzyme activity using the substrate phosphatidylcholine ,the results demonstrated that the recombinant PLA2 protein of PfDNV was successfully expressed, which would facilitate the preparation of the antiserum and the further study of the biological properties of PLA2 enzyme.更多还原