HPV11E7-TpN47融合基因原核表达系统的构建.DOCVIP

HPV6bL1- TpN15 嵌合基因原核表达系统的构建 郑丹1，楼迪栋2，屈植风,3，刘菁4，周翠萍5 贵阳市妇幼保健院保健部（郑丹，屈植风,，刘菁，周翠萍）；贵阳医学院法医学教研室（楼迪栋） 贵州 贵阳 550003 [摘要] 目的 分别从梅毒螺旋体(Treponema pallidum，Tp)尖锐湿疣CA)组织中扩增人乳头瘤病毒(HPV)型TpN15基因，- TpN15 嵌合基因及其原核表达系统。方法 PCR 分别从CA 组织Tp临床菌株中扩增HPV6b L1和TpN15 基因片段，构建HPV6b L1- TpN15嵌合基因，将其克隆入原核表达载体pET32a(+)构建重组质粒pET32a(+)/- TpN15，经测序鉴定后转化大肠杆菌BL21（DE3），经异丙基硫代-β-D-半乳糖苷IPTG)诱导后表达的融合蛋白用SDS）-聚丙烯酰胺凝胶电泳（PAGE）、蛋白质印迹法分析鉴定 结果 - TpN15融合蛋白在原核表达系统中得到了较高表达。 结论 原核表达的- TpN15融合蛋白具较强的抗原性。 The construction of prokaryotic expr ession product of HPV6b L1- TpN15 fusion gene ZHENG Dan11，Lou Di-dong2，Qu Zhi-feng3，Liu-Jing4，Zhou Cui-ping5 (Department of Maternal and Child Health Hospital Conservancy Branch of Guiyang City，Guizhou Guiyang 550003，China ) 【Abstract】 Objective To amplify and TpN15 gene from DNAs of a clinical strain Treponema pallidum and Escherichia coli strain，respectively，and construct- TpN15fusion gene and its prokaryotic expression system. Methods The gene，full-length，encoding for HPVTpN15 protein was amplified by PCR，and cloned into vector pET32a(+) to form recombinant pET32a(+)/HPV TpN15 plasmid，then transformed into the E.Coli BL21(DE3). The His- TpN15 fusion protein was expressed when induced by adding IPTG ，purified with Ni-NTA Agarose affinity column and analyzed by SDSand Western blotting. Results The recombinant plasmid was identified and confirmed with enzyme digestion and sequencing. A high level expression of His- HPV6b L1- TpN15 fusion protein was obtained and purified successfully. Conclusion The HPV6b L1- TpN15fusion protein possessed strong antigenicity. 【Key words】 Condyloma acuminate，Treponema pallidum; fusion protein; Cloning; expression 梅毒是一种临床表现极为复杂、几乎可累及全身各器官的性病。我国梅毒发病率逐年递 作者 增。梅毒的病原体是梅毒螺螺旋体(Treponema pallidum，Tp) ，国外已有研究显示，TpNl5TpN17、TpN44.5、TpN47 等外膜蛋白均可用于Tp的检测，TpN15为主要外膜蛋白，是Tp的特异性表面抗原[1]，且具有高免疫原性。因TpN15基因核苷酸序列高度保守，Tp基因工程疫苗的侯选抗原。而由人乳头瘤病毒(human papillomavirus，HPV)6、1l型等低危型别引起的尖锐湿疣，在我国发病呈逐年上升趋势，目前仍缺乏有效的防治措施。HPV和TP均可侵犯生殖道黏膜致病，TP感染可损伤局部黏膜，增加HPV感染机会。因此