| 摘要: |
| 【目的】获得猪囊尾蚴凋亡蛋白酶抑制剂（API）基因在大肠杆菌中的最佳表达条件，为API功能研究奠定基础。【方法】以表达载体pEGX-4T-1、pET-30a及猪囊尾蚴API基因的原核表达载体pEGX-4T-1/API-Flag、pET-30a/API为材料，对影响API蛋白表达的载体（pEGX-4T-1、pET-30a）、温度（25，30，37 ℃）、诱导时间（2，4，6，8 h）、IPTG终浓度（0.2，0.4，0.6，0.8，1.0 mmol/L）和卡那霉素终浓度（100，200，300，400 mmol/L）等条件进行优化，筛选重组蛋白最佳的表达条件。对在最佳条件下获取的目的蛋白进行超声处理，4 ℃、10 000 r/min离心分别收集上清液和沉淀，对目的蛋白进行可溶性分析；SDS-PAGE后，将目的蛋白条带转印至硝酸纤维素膜上，与抗His标签抗体共孵育，检测目的蛋白的反应原性。【结果】 当选择pET系列载体（pET 30a）时，API表达量高于其在pGEX系列（pGEX-4T-1）中的表达量。API融合蛋白最佳表达条件为：在含200 mmol/L卡那霉素的LB培养基上于37 ℃培养3 h后，加入终浓度为0.4 mmol/L的IPTG，再于30 ℃诱导培养6 h。SDS-PAGE结果表明，pET-30a/API融合蛋白分子质量约为53 ku，与预期蛋白分子质量一致。Western blot检测结果显示，重组蛋白pET 30a/API能够识别特异性抗体，显示获得了大量表达正确的目的蛋白。【结论】 确定了API基因的最佳表达条件，获得了较大表达量的API融合蛋白。 |
| 关键词: 猪囊尾蚴 凋亡蛋白酶抑制基因 API蛋白 原核表达 |
| DOI： |
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| 基金项目:家畜疫病病原生物学国家重点实验室开放基金项目（SKLVEB2014KFKT007）；河南科技学院高层次人才科研项目（2013008） |
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| Optimization of prokaryotic expression conditions for apoptosis protease inhibitor gene from Cysticercus cellulosae |
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SU Wenqiang,HU Xinyu,WANG Qiuxia,et al
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| Abstract: |
| 【Objective】This study obtained the optimal expression conditions of apoptosis protease inhibitor (API) gene of Cysticercus cellulosae in Escherichia coli to provide basis for studying its functions.【Method】The factors influencing API expression including expression vector (pEGX-4T-1 and pET-30a),temperature (25，30，and 37 ℃),inducing time (2，4，6，and 8 h),final concentration of inductor IPTG (0.2，0.4，0.6，0.8，and 1.0 mmol/L) and kanamycin (Kan) (100，200，300，and 400 mmol/L) were optimized.The obtained protein under optimal conditions was treated by ultrasonic and centrifuged under 4 ℃ at the rate of 10 000 r/min.Supernatant and precipitation were collected for soluble analysis.After SDS-PAGE,the target protein bands were transferred to NC membrane and the reactivity was detected after incubation with anti His tag antibody.【Result】The pET30a was better than pGEX-4T-1 to express the API protein.The optimal expression conditions were 200 mmol/L Kan,LB medium,and 3 h culture at 37 ℃ before adding 0.4 mmol/L IPTG and 6 h culture at 30 ℃.SDS-PAGE analysis showed that the size of pET30a/API fusion protein was 53 ku.Western blot analysis indicated that the anti His monoclonal antibody could specifically bound to pET30a/API fusion protein.【Conclusion】This study obtained the optimal expressed conditions of APIgene. |
| Key words: Cysticercus cellulosae apoptosis protease inhibitor gene API protein prokaryotic expression |
