副溶血弧菌外膜铁蛋白受体pvuA基因的原核表达及产物的诱导条件优化

Prokaryotic Expression of Outer Membrane Ferritin Receptor pvuA Gene of Vibrio parahaemolyticus and Optimization of Induction Conditions for Product

为构建弧菌铁蛋白受体pvuA重组质粒,提高其在大肠杆菌BL21中的表达产量,优化表达条件,并为其免疫原性研究奠定基础,从副溶血弧菌基因组DNA扩增了弧菌铁蛋白受体pvuA基因,构建了重组质粒pET-28a(+)-ferric vibrioferrin receptor,转入大肠杆菌BL21并经异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达蛋白。在单因素试验的基础上,以菌体初始浓度、诱导时间、诱导温度、诱导剂浓度为自变量,菌体蛋白浓度为响应值,根据响应面法的Box-Benhnken中心设计原理,研究自变量及其交互作用对弧菌铁蛋白产量的影响,利用Design-Expert和响应面分析相结合的方法对诱导条件进行优化。IPTG诱导获得的重组蛋白以包涵体的形式存在,优化后最终确定重组弧菌铁蛋白受体pvuA最佳表达条件为菌体初始浓度OD600=0.6,诱导时间10 h,诱导温度37℃,IPTG浓度为1.0 mmol·L^(−1),此时包涵体沉淀中蛋白含量最高,为11.00 mg·mL^(−1)。构建了弧菌铁蛋白受体pvuA的大肠杆菌重组表达质粒,通过优化表达条件提高了纯化蛋白产率,为研究弧菌铁蛋白受体蛋白的多克隆抗体的制备和应用奠定了基础。

To obtain the recombinant plasmid of ferric vibrioferrin receptor pvuA and improve the expression yield of pvuA in Escherichia coli BL21,optimize the expression conditions,and lay a foundation for its immunogenicity study,the ferric vibriofer⁃rin receptor gene pvuA was amplified from the genomic DNA of Vibrio parahaemolyticus.The recombinant plasmid pET-28a(+)-ferriferrin receptor was constructed,transformed into E.coli BL21,and induced by isopropylβ-D-thiogalactoside(IPTG).Based on the single factor experiment,the effects of independent variables and their interaction on the production yield of ferric vibrio⁃ferrin receptor were studied by response surface methodology.The independent variables were initial cell concentration,induc⁃tion time and induction temperature,inducer concentration,and the response value was the yield of bacterial protein.The induc⁃tion conditions were optimized by the combination of Design Expert and response surface analysis.The ferric vibrioferrin receptor was expressed as inclusion bodies.The optimal expressioninitial cell concentration was OD600=0.6,temperature was 37℃,induction time was 10 h,IPTG concentration was 1.0 mmol·L^(−1).At this time,the protein content of inclusion bodies precipitation was the highest(11.00 mg·mL^(−1)).The E.coli recombinant plasmid of ferric vibrioferrin receptor pvuA was constructed,the yield of purified protein was improved by optimizing the expression conditions,which laid a foundation for the preparation and applica⁃tion of polyclonal antibody against Vibrio spp..