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目的为铜绿假单胞菌外膜蛋白H(OprH)工业发酵与疫苗开发研究奠定基础。方法采用分子克隆技术构建OprH蛋白的表达菌株,通过正交试验设计筛选OprH菌株的最佳培养条件与表达条件。利用切胶纯化获得OprH蛋白并免疫小鼠,制备OprH蛋白多克隆抗体,采用ELISA法检测抗体滴度,蛋白质印迹法检测抗血清特异性;对小鼠免疫OprH蛋白,攻毒铜绿假单胞菌,检测蛋白的免疫保护作用。结果 OprH重组载体双酶切、DNA测序鉴定结果,以及蛋白表达、纯化条带大小与预测一致。OprH菌株最佳培养条件为转速230r/min,葡萄糖浓度0%,装液量50mL;最佳诱导表达条件为异丙基-β-D-硫代半乳糖苷终浓度0.3mmol/L,诱导时菌液D600值0.8,温度32℃,诱导时间3h。ELISA法获得OprH抗血清滴度达1∶1 600,蛋白质印迹法证实抗血清具有很好的特异性。OprH免疫小鼠激活的特异性免疫对小鼠铜绿假单胞菌感染的保护率达到46.15%,与对照相比较差异有统计学意义(P<0.05)。结论成功构建OprH表达载体,纯化获得OprH蛋白,制备OprH蛋白多克隆抗体,实验结果表明OprH蛋白对小鼠铜绿假单胞菌感染具有免疫保护作用,并获得OprH蛋白菌株的最佳培养条件与表达条件。
Objective To lay the foundation for industrial fermentation and vaccine development of Pseudomonas aeruginosa outer membrane protein H (OprH). Methods OprH protein was constructed by molecular cloning technique. The optimal culture conditions and expression conditions of OprH were screened by orthogonal design. OprH protein was purified by gel-cutting and immunized to prepare polyclonal antibody against OprH protein. Antibody titers were detected by ELISA and antisera were detected by Western blotting. Immunization of mice with OprH protein and challenge with P. aeruginosa , Detect the protein’s immune protection. Results OprH recombinant vector double digestion, DNA sequencing identification results, and protein expression, purification band size and prediction. The optimal culture conditions of OprH strain were 230r / min of rotation speed, 0% of glucose concentration and 50mL of liquid volume. The optimal induction conditions were 0.3mmol / L of isopropyl-β-D-thiogalactopyranoside Bacteria D600 value 0.8, temperature 32 ℃, induction time 3h. The titer of OprH antiserum was 1: 1 600 by ELISA, and the antiserum was proved to have good specificity by Western blotting. The rate of protection against Pseudomonas aeruginosa infection in mice immunized with OprH was 46.15%, which was significantly different from the control (P <0.05). Conclusion OprH expression vector was successfully constructed and OprH protein was purified to prepare polyclonal antibody of OprH protein. The experimental results showed that OprH protein had protective immunity against Pseudomonas aeruginosa infection in mice, and the best culture conditions and expression of OprH protein strain condition.