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目的构建并鉴定铜绿假单胞菌重组质粒pGEX-OprF,研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法以铜绿假单胞菌PAOl标准株总RNA为模板,自行设计引物,采用RT-PCR方法扩增OprF抗原编码基因,经酶切、连接后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,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 RT-PCR扩增出1 016 bp的OprF编码基因;重组质粒经双酶切和测序鉴定证实OprF基因成功插入pGEX-1λT中;SDS-PAGE分析表达产物分子质量单位约为61 ku,与预期结果一致,表达的蛋白质占菌体总蛋白的16%;Western blot鉴定重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别。结论成功构建了铜绿假单胞菌重组质粒pGEX-OprF,该质粒在E.coliBL21(DE3)中高效融合表达,表达的融合蛋白具有抗原特异性。
Objective To construct and identify the recombinant plasmid pGEX-OprF of Pseudomonas aeruginosa and study its expression in Escherichia coli BL21 (DE3). Methods The total RNA of Pseudomonas aeruginosa PAO1 strain was used as a template to design the primers. The gene encoding OprF antigen was amplified by RT-PCR and cloned into Escherichia coli-Bifidobacterium shuttle expression The recombinant plasmid pGEX-OprF was constructed and transformed into E.coli BL21 (DE3) competent cells. After induced by IPTG, The expression products were analyzed and identified. Results The 1 016 bp OprF gene was amplified by RT-PCR. OprF gene was successfully inserted into pGEX-1λT by double enzyme digestion and sequencing. The molecular mass unit of the expressed product was about 61 ku by SDS-PAGE, The results were consistent, the expression of protein accounted for 16% of the total bacterial protein; Western blot identification of recombinant protein Pa outer membrane antigen can be immune to mouse sera. Conclusion The recombinant plasmid pGEX-OprF of Pseudomonas aeruginosa was successfully constructed. The plasmid was highly expressed in E. coli BL21 (DE3) and the expressed fusion protein was antigen-specific.