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

Construction and eukaryotic expression of a recombinant plasmid encoding Pseudomonas aeruginosa toxA and type Ⅲ secretion system pcrV

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。

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 .