Esat6-卡介苗重组疫苗的构建免疫原性及抗结核作用的初步研究

The study on construction immunogenicity protection against M.tuberculosis of esat6-recombinant BCG vaccine

目的构建结核分枝杆菌esat6-卡介苗重组疫苗,研究其免疫原性及抗结核作用,以期获得结核病预防性或治疗性疫苗。方法通过基因工程重组技术将结核分枝杆菌保护性抗原esat6的编码基因与穿梭质粒载体pYUB295重组,采用电穿孔技术导入卡介苗中,应用聚合酶链反应(PCR)扩增、聚丙烯酰胺凝胺电泳(PAGE)鉴定重组卡介苗。通过酶联免疫吸附试验(ELISA)法检测其血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数。通过观察esat6-卡介苗重组疫苗对结核分枝杆菌感染的预防试验中实验动物半数死亡时间、一定时间内的死亡率、大体病变、T细胞及B细胞免疫功能等指标评价esat6-卡介苗重组疫苗对结核分枝杆菌感染的预防效果。结果PCR扩增、限制性内切酶酶切、DNA测序鉴定、PAGE电泳表明成功地构建了esat6基因pYUB295重组质粒,ESAT6蛋白在卡介苗中分泌表达。重组卡介苗免疫原性试验表明:esat6-卡介苗重组疫苗组有ESAT6特异性抗体产生,45d时达到最高水平。脾淋巴细胞增殖试验表明各组刺激指数均达2.0以上,esat6重组卡介苗免疫组小鼠脾淋巴细胞的刺激指数稍高于对照组。预防试验表明:esat6-卡介苗重组疫苗组、卡介苗组与生理盐水对照组比都能延长结核分枝杆菌感染小鼠的半数死亡时间,降低2个月内的死亡率。其中esat6-卡介苗重组疫苗组效果显著,与卡介苗组比有显著差异。结核分支杆菌攻击后2个月,处死小鼠时,小鼠脏器大体病变及脏器培养结果表明:esat6-卡介苗重组疫苗免疫组小鼠比其他各组小鼠结核菌的负荷轻。抗体检测结果及淋巴细胞增殖实验各组结果差异无统计学意义。结论正确构建了esat6-卡介苗重组疫苗,esat6-卡介苗重组疫苗能提高卡介苗对结核分枝杆菌感染的预防作用。

Objective Construct esat6-recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively. Methods The gene of esat6 was amplified by PCR and recomhined with shuttle plasmids pYUB295. The recombinant shuttle plasmid was identified by PCR,enzyme digestion and DNA sequen- cing and then transformed into BCG by electroporation. The antigen-specific antibody levels of mouse immunized with recombinant BCG were evaluated by ELISA and multiplication of mouse lymph--cell was detected by MTS. The protection against M. tuberculosis of esat6-recombinant BCG vaccine were tested by their prevention and treatment experiments. Results PCR,enzyme digestion,DNA sequencing and SDS-PAGE results showed：The esat6- recombinant BCG vaccine was constructed successfully and can express extrinsic esat6 gene. The immunity experiment showed：extrinsic gene can be express in BCG and can stimulate B cell of mouse to produce antibody, the antibody level reached the peak after 45 days; The lymph-cells of each group proliferated when stimulated by different antigen, all stimulation index reached 2, The stimulation index of each group had no obviously difference except esat6-recombiant BCG vaccine group. The prevention experiment against M. tuberculosis indicted ..esat6- recombinant BCG vaccine and BCG can extend mean time to death and reduce death rate in 2 month of those mouse, the protection effect of esat6-recombinant BCG vaccine was more effective than BCG. Conclusion The esat6-recombinant BCG vaccine was constructed successfully and can prevent TB effectively than BCG.