人乳头瘤病毒6b型重组减毒沙门菌的构建及表达

目的：构建ＨＰＶ６ｂ　Ｌ１Ｅ７嵌合基因重组减毒沙门菌，为进一步粘膜免疫奠定基础。方法：用ＰＣＲ方法扩增获得ＨＰＶ６ｂ　的　Ｌ１Ｅ７　嵌合基因片段，ＴＡ　克隆后，再将Ｌ１－Ｅ７　基因克隆入ｐＴＥＴｎｉｒ１５　沙门菌表达质粒。用电转化法将重组表达质粒导入鼠伤寒减毒沙门菌Ｓ．ＢＲＤ５０９（△ａｒｏＡ，△ａｒｏＣ）中，厌氧诱导其表达，蛋白样品做ＳＤＳ－ＰＡＧＥ　凝胶电泳　和ｗｅｓｔｅｒｎ　ｂｌｏｔ分析。结果：用ＢａｍＨＩ和ＳａｌＩ双酶切重组表达质粒ｐＴＥＴｎｉｒ１５６ｂＬ１Ｅ７可释放２３８９ｂｐ和１５５４ｂｐ两片段，结果与预计相符。ｗｅｓｔｅｒｎ　ｂｌｏｔ结果显示，重组沙门菌在约５６ＫＤ处有明显特异蛋白条带，而对照菌则无。结论：该重组沙门菌能特异地表达ＨＰＶ６ｂ　Ｌ１－Ｅ７　融合蛋白，人乳头瘤病毒ＨＰＶ６ｂ　Ｌ１－Ｅ７重组减毒沙门菌构建成功。

Construction of a recombinant attenuated salmonella strains expressing the human papillomavirus type 6bL1-E7 proteins

Objective:To construct a recombinant attenuated s almonella typhimurium S.BRD509 strains expressing the human papillomavirus type 6bL1 E7 proteins , so that it can immunize mucosally and elicit humoral, secret ory and cell mediated immune responses.Methods:HPV L1 E7 cheme ric gene was amplyfied by PCR and cloned in a salmonella experessing plasmid na med pTETnir15. After induced anaerobically, the expressed protein was detected b y SDS PAGE and Western blot analysis.Results:The recombinant 6bL1 E7 salmonella showed expression of the L1 E7 gene as a fusion protein of 56KD while the control remained negative.Conclusion:The re combinant attenuated salmonella of human papillomavirus type 6bL1 E7,has been successfully constructed,and the L1 E7 fusion protein can be expressed in vitro.