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°C)、诱导时间(2,4,6,8h)、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°C、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°C培养3h后,加入终浓度为0.4 mmol/L的IPTG,再于30°C诱导培养6h.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.%[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 °C),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 °C at the rate of 10 000 r/min.Supernatant and precipitation were collected for soluble analysis.After SDSPAGE,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 °C before adding 0.4 mmol/L IPTG and 6 h culture at 30 °C.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 API gene.
