医学研究生学报
醫學研究生學報
의학연구생학보
JOURNAL OF MEDICAL POSTGRADUATE
2014年
7期
694-697
,共4页
铜绿假单胞菌%外毒素A%核酸疫苗%pcrV基因
銅綠假單胞菌%外毒素A%覈痠疫苗%pcrV基因
동록가단포균%외독소A%핵산역묘%pcrV기인
Pseudomonas aeruginosa%Exotoxin A%DNA vaccine%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。
目的：外毒素A（ toxA基因編碼）是銅綠假單胞菌毒力最彊的因子之一，PcrV（ pcrV基因編碼）是銅綠假單胞菌Ⅲ型分泌繫統的關鍵調控因子之一。文中旨在構建重組toxA和pcrV基因的銅綠假單胞菌覈痠疫苗，併在HEK-293細胞中錶達目的蛋白。方法 PCR法從銅綠假單胞菌基因組基因中擴增齣 toxA和pcrV基因，點突變法對toxA基因進行減毒優化，隨後將突變的toxAm基因和pcrV基因分彆插入pIRES真覈錶達質粒的2箇多剋隆位點，構建真覈雙錶達重組質粒pIRES-toxAm-pcrV。脂質體法將pIRES-toxAm-pcrV瞬時轉染入HEK-293細胞，通過Western blot檢測toxAm及pcrV在真覈細胞中的錶達。結果構建的真覈錶達質粒pIRES-toxAm-pcrV，經脂質體轉染HEK-293細胞後，在其細胞內檢測到目的蛋白的錶達。結論成功構建pIRES-toxAm-pcrV錶達載體併在轉染的真覈細胞中得以有效的錶達，為研究銅綠假單胞菌預防性疫苗奠定瞭實驗基礎。
목적：외독소A（ toxA기인편마）시동록가단포균독력최강적인자지일，PcrV（ pcrV기인편마）시동록가단포균Ⅲ형분비계통적관건조공인자지일。문중지재구건중조toxA화pcrV기인적동록가단포균핵산역묘，병재HEK-293세포중표체목적단백。방법 PCR법종동록가단포균기인조기인중확증출 toxA화pcrV기인，점돌변법대toxA기인진행감독우화，수후장돌변적toxAm기인화pcrV기인분별삽입pIRES진핵표체질립적2개다극륭위점，구건진핵쌍표체중조질립pIRES-toxAm-pcrV。지질체법장pIRES-toxAm-pcrV순시전염입HEK-293세포，통과Western blot검측toxAm급pcrV재진핵세포중적표체。결과구건적진핵표체질립pIRES-toxAm-pcrV，경지질체전염HEK-293세포후，재기세포내검측도목적단백적표체。결론성공구건pIRES-toxAm-pcrV표체재체병재전염적진핵세포중득이유효적표체，위연구동록가단포균예방성역묘전정료실험기출。
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 .
