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)临床菌株和尖锐湿疣(CA)组织中扩增人乳头瘤病毒(HPV)6b型L1和TpN15基因，构建HPV6b L1- TpN15 嵌合基因及其原核表达系统。方法 采用PCR 分别从CA 组织及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(+)/HPV6b L1- 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十二烷基磺酸钠（SDS）-聚丙烯酰胺凝胶电泳（PAGE）、蛋白质印迹法分析鉴定。 结果 HPV6b L1- 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HPV6b L1- TpN15融合蛋白具较强的抗原性。 [关键词]尖锐湿疣，梅毒；融合蛋白；克隆；表达 [中图分类号] R392 [文献标识号] A [文章编号] 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 HPV6b L1 and TpN15 gene from DNAs of a clinical strain Treponema pallidum and Escherichia coli strain，respectively，and construct HPV6b L1- TpN15fusion gene and its prokaryotic expression system. Methods The gene，full-length，encoding for HPV6b L1and TpN15 protein was amplified by PCR，and cloned into vector pET32a(+) to form recombinant pET32a(+)/HPV6b L1- TpN15 plasmid，then transformed into the E.Coli BL21(DE3). The His- HPV6b L1- 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】 Condylomata 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 作者简介：郑丹（1977-），女，仡佬族，贵州，主治医师，硕士，从事妇女保健工作。 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，T