猪囊尾蚴特异性抗原cC1重组耻垢分枝杆菌疫苗的构建与鉴定

发布时间：2019-06-09 23:03

【摘要】：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。
[Abstract]:Objective to construct a recombinant Mycobacterium pubis vaccine expressing cC1 antigen of cysticercus cellulosae. Methods pET28a-cC1 plasmid DNA, was extracted by xho I and BamH I double enzyme digestion, xho I was digested with Klenow enzyme, pMV261 shuttle plasmid vector was extracted at the same time, Hind III and BamH I were digested with Hind III and BamH I, and Hind III was digested with Klenow enzyme. The purified enzyme product was ligated by T _ 4 ligase to construct pMV261-cC1 shuttle plasmid. After sequencing, the pMV261cC1 shuttle plasmid was transformed into Mycobacterium pubis by electroporation, and the recombinant Mycobacterium pubis was constructed. After heat induction, The serum of patients with cysticercosis was identified by Western-blotting. In addition, the growth state of normal Mycobacterium pubis and recombinant cC1 was compared and the growth curve was drawn. Results the recombinant shuttle vector was digested by enzyme and sequenced. After heat induction, SDS-PAGE electrophoresis analysis showed that exogenous protein was expressed at 40 kDa, which was consistent with the expected value. Western-blotting showed that it could be recognized by serum of patients with cysticercosis. The results showed that cC1 antigen gene was successfully expressed in Mycobacterium pubis. There was no significant difference in proliferation characteristics between recombinant Mycobacterium pubis and normal Mycobacterium pubis. Conclusion Recombinant Mycobacterium pubis vaccine expressing cysticercus specific antigen cC1 was successfully constructed.
【作者单位】：蚌埠医学院病原生物学教研室;安徽省感染与免疫重点实验室;蚌埠医学院免疫学教研室;
【基金】：国家自然科学基金(30600518/C030112) 安徽省高等学校省级自然科学研究重点项目(KJ2013A185)
【分类号】：R392

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