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目的构建表达猪囊尾蚴cC1抗原的重组耻垢分枝杆菌疫苗。方法提取pET28a-cC1质粒DNA,用xho I和BamH I双酶切,用Klenow酶补平xho I酶切末端,同时提取pMV261穿梭质粒载体,用Hind III和BamH I双酶切,用Klenow酶补平Hind III酶切末端,纯化后的酶切产物通过T4连接酶连接,构建pMV261-cC1穿梭质粒,测序验证正确后,将pMV261cC1穿梭质粒经电穿孔法转化耻垢分枝杆菌,构建重组耻垢分枝杆菌,经热诱导后,以猪囊尾蚴病患者血清为一抗进行Western-blotting鉴定。此外,比较正常耻垢分枝杆菌和重组cC1耻垢分枝杆菌生长状态并绘制生长曲线。结果构建的重组穿梭载体经酶切、测序验证正确。构建的重组耻垢分枝杆菌经热诱导后,SDS-PAGE电泳分析显示,在40 kDa处有外源性蛋白表达,与预期值相符。Western-blotting显示其可被猪囊尾蚴病患者血清识别,表明cC1抗原基因在耻垢分枝杆菌中表达成功。重组耻垢分枝杆菌与正常耻垢分枝杆菌的增殖特性无明显差异。结论成功构建了表达猪囊尾蚴特异性抗原cC1的重组耻垢分枝杆菌疫苗。
Objective To construct a recombinant Mycobacterium smegmatis vaccine expressing cC1 antigen of Cysticercus cellulosae. Methods The pET28a-cC1 plasmid DNA was extracted and double-digested with xho I and BamH I. The digested end of xho I was filled in with Klenow enzyme and pMV261 shuttle plasmid vector was digested with Hind III and BamH I, The end of Hind III digestion and the purified digested product were ligated by T4 ligase to construct the shuttle plasmid pMV261-cC1. After sequencing and verification, pMV261cC1 shuttle plasmid was transformed into M. smegmatis by electroporation to construct recombinant smegma Mycobacterium, after heat induction, the serum of cysticercosis patients as the first antibody Western-blotting identification. In addition, we compared the growth status of normal M. smegmatis and recombinant cC1 M. smegmatis and plotted the growth curve. Results The recombinant shuttle vector was verified by sequencing and sequencing. The recombinant Mycobacterium smegmatis was induced by heat, SDS-PAGE electrophoresis analysis showed that exogenous protein at 40 kDa was in line with the expected value. Western-blotting showed that it could be recognized by the serum of patients with cysticercosis, indicating that cC1 antigen gene was successfully expressed in Mycobacterium smegmatis. The proliferation of recombinant Mycobacterium smegmatis and normal M. smegmatis showed no significant difference. Conclusion The recombinant Mycobacterium smegmatis vaccine expressing cC1 antigen of C. cysticercus was successfully constructed.