| 摘要: |
| 【目的】构建溶藻弧菌外膜蛋白OmpK原核表达载体，优化OmpK蛋白的诱导表达条件，制备OmpK蛋白小鼠多克隆抗体。【方法】通过PCR扩增获得ompK基因，连接pET-32a质粒构建OmpK-pET32a载体，将其转化大肠杆菌BL21后进行诱导表达。利用切胶纯化获得OmpK蛋白后，免疫小鼠制备OmpK蛋白多克隆抗体，采用ELISA法检测抗体效价，Western-Blotting法检测抗血清特异性。通过正交试验，获得OmpK菌株的最佳诱导表达条件与培养条件。【结果】PCR扩增获得了长816 bp的ompK基因；成功构建了OmpK-pET32a载体，其诱导表达产物分子质量为30 ku，与预期结果一致。ELISA法获得OmpK抗血清效价达1∶1 600，Western-Blotting试验证实抗血清具有很好的特异性。OmpK菌株最佳诱导表达条件为：菌液OD600值0.8，IPTG终浓度0.3 mmol/L，诱导时间8 h，诱导温度32 ℃；最佳培养条件为：不添加葡萄糖，转速230 r/min，装液量50 mL。【结论】成功制备了OmpK蛋白多克隆抗体，确定了OmpK蛋白菌株的最佳诱导表达条件与培养条件。 |
| 关键词: 溶藻弧菌 OmpK蛋白 原核表达 多克隆抗体 |
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| 基金项目:陕西省教育厅科学研究计划项目(2013JK0723)；陕西理工学院人才启动项目(SLGQD13-15) |
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| Prokaryotic expression and polyclonal antibody preparation of outer membrane protein OmpK in Vibrio alginolyticus |
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CHEN Chun-lin,LIU Xiang,JU Xiong
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| Abstract: |
| 【Objective】This study aimed to construct prokaryotic expression vector of Vibrio alginolyticus outer membrane protein K (OmpK),optimize the expression condition of OmpK,and prepare the polyclonal antibodies of mouse anti-OmpK.【Method】ompK gene was obtained by PCR amplification before being connected to pET-32a plasmid for OmpK-pET32a vector.Then,the vector was transformed to E.coli BL21 for OmpK protein expression strain.OmpK was purified by the gel slicing method,and the polyclonal antibody was prepared using immunized mice.The antibody titer and specificity were detected by ELISA and Western-Blotting,respectively.The optimal inducing and culturing conditions were also gained by orthogonal experiment.【Result】PCR amplification obtained 816 bp ompK gene.OmpK-pET32a vector was constructed successfully,and the induced expression product was 30 ku.The OmpK antibody titer was 1∶1 600 obtained by ELISA,and Western Blotting proved that the antiserum had good specificity.The optimal inducing conditions for OmpK protein expression were:strain OD600 value,IPTG final concentration,inducing time and temperature were 0.8,0.3 mmol/L,8 h,and 32 ℃,respectively while the optimal culturing conditions were:rotation rate,glucose concentration,and medium volume were 230 r/min,0 glucose and 50 mL,respectively.【Conclusion】The OmpK polyclonal antibodies were successfully prepared and the optimal inducing and culturing conditions were obtained. |
| Key words: Vibrio alginolyticus OmpK protein prokaryotic expression polyclonal antibody |
