【摘要】 目的构建和鉴定铜绿假单胞菌重组双歧杆菌(rBb)-OprF疫苗。方法以铜绿假单胞菌PAOl标准株提取总RNA为模板,自行设计引物,RT-PCR扩增获得OprF抗原编码基因,经酶切、连接定向克隆入大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-OprF。转化E.coliBL21(DE3)感受态细胞,抽提质粒行酶切电泳和测序验证后,电穿孔转化到Bb中,构建铜绿假单胞菌rBb-OprF疫苗,抽提质粒进行PCR扩增鉴定。结果 RT-PCR扩增出1 016bp的OprF编码基因;重组质粒经双酶切鉴定,切出4 947bp的载体片段和10 16bp的目的基因片段;以rBb抽提质粒为模板进行PCR扩增可得到1 016bp的oprF基因片段。结论成功构建了铜绿假单胞菌rBb-OprF疫苗,为该疫苗的进一步研究奠定基础。更多还原

【Abstract】 In order to construct and identify recombinant Bifidobacteria(rBb)-OprF vaccine of Pseudomonas aeruginosa,the total RNA was extracted from Pseudomonas aeruginosa(PAO1),and used as template.The OprF coding gene was amplified by RT-PCR,digested and then cloned into E.coli-Bifidobacteria shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-OprF.The recombinant plasmid was transformed into E.coli BL21(DE3) and screened.The product was identified by sequencing and electrophoresis,and then introduced into Bb by electroporation to construct rBb-OprF vaccine.The vaccine was identified by PCR.The results showed that OprF coding gene was successfully amplified by PCR and the products of restriction endonuclease digestion were the same as expected(1016bp oprF gene and 4947bp pGEX-1λT).The oprF gene fragment was amplified by PCR from the template of pGEX-OprF extracted from rBb vaccine.The recombinant Bb-OprF vaccine of Pseudomonas aeruginosa was successfully constructed.The results could provide for the basis to further research of rBb-OprF vaccine.更多还原