重组铜绿假单胞菌外毒素A和pcrV基因质粒的构建及真
核表达
姜明子;冯旰珠
【期刊名称】《医学研究生学报》 【年(卷),期】2014(000)007
【摘要】Objective Exotoxin A ( encoded by gene toxA ) , one of the most toxic protein secreted by pseudomonas aerugi-nosa(P.a.), and PcrV (encoded by gene pcrV), key component to type Ⅲsecretion system of P.a., both matter significantly to the virulence of P.a.The article was to construct a novel DNA vaccine encoding a mutated toxA gene and the pcrV gene of P .a.and i-dentify gene expressions in eukaryotic cells . Methods The genes of toxA and pcrV were amplified by PCR , and the toxA gene was mutated to reduce the toxicity of Exotoxin A .Then gene fragments toxA m and pcrV were inserted into eukaryotic expression plasmid pIRES simultaneously to construct a recombinant DNA vaccine pIRES-toxAm-pcrV.The novel plasmid was transfected into HEK-293 cells by lipofectamine 2000 .The expressions of toxA m and pcrV were detected by Western blot . Results Gel electrophoresis demon-strated the target gene fragments encoding Exotoxin A and PcrV .Western blot exhibited proteins encoded toxA and pcrV expressed by HEK 293 cells. Conclusion
The
recombinant
plasmid
pIRES-toxAm-pcrV
was
successfully constructed .Western blot analysis indi-cated the
expressions of toxA m and pcrV in HEK-293 cells.It may be used as a potential candidate of preventive vaccine of Pseudo-monas aeruginosa .%目的：外毒素A（ toxA基因编码）是铜绿假单胞菌毒力最强的因子之一，PcrV（ pcrV基因编码）是铜绿假单胞菌Ⅲ型分泌系统的关键调控因子之一。文中旨在构建重组toxA和pcrV基因的铜绿假单胞菌核酸疫苗，并在HEK-293细胞中表达目的蛋白。方法 PCR法从铜绿假单胞菌基因组基因中扩增出 toxA和pcrV基因，点突变法对toxA基因进行减毒优化，随后将突变的toxAm基因和pcrV基因分别插入pIRES真核表达质粒的2个多克隆位点，构建真核双表达重组质粒pIRES-toxAm-pcrV。脂质体法将pIRES-toxAm-pcrV瞬时转染入HEK-293细胞，通过Western blot检测toxAm及pcrV在真核细胞中的表达。结果构建的真核表达质粒pIRES-toxAm-pcrV，经脂质体转染HEK-293细胞后，在其细胞内检测到目的蛋白的表达。结论成功构建pIRES-toxAm-pcrV表达载体并在转染的真核细胞中得以有效的表达，为研究铜绿假单胞菌预防性疫苗奠定了实验基础。 【总页数】4页(694-697)
【关键词】铜绿假单胞菌;外毒素A;核酸疫苗;pcrV基因 【作者】姜明子;冯旰珠
【作者单位】210011 南京，南京医科大学第二附属医院呼吸内科;210011 南京，南京医科大学第二附属医院呼吸内科 【正文语种】中文 【中图分类】R563 【文献来源】
https://www.zhangqiaokeyan.com/academic-journal-cn_journal-medical-postgraduates_thesis/0201252347368.html 【相关文献】
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重组铜绿假单胞菌外毒素A和pcrV基因质粒的构建及真核表达
