mpt64-卡介苗重组疫苗的构建、免疫原性及抗结核作用

The Study On Construction,Immunogenicity and Protection Against Mycobacterium tuberculosis of mpt64-recombinant BCG Vaccine

通过基因工程重组技术将结核分枝杆菌保护性抗原MPT64的编码基因与穿梭质粒载体pYUB295重组,采用电穿孔技术将重组质粒导入到卡介苗中,应用聚合酶链反应(PCR)扩增、聚丙烯酰胺凝胶电泳(PAGE)对mpt64-卡介苗重组疫苗鉴定:成功地构建了MPT64基因pYUB295重组质粒,MPT64蛋白在卡介苗中能分泌表达。卡介苗重组疫苗免疫原性试验表明:只有mpt64-卡介苗重组疫苗组有MPT64特异性抗体产生,45天时达到最高水平。脾淋巴细胞增殖试验表明各组小鼠平均刺激指数达2.0以上,mpt64-卡介苗重组疫苗组小鼠脾淋巴细胞的刺激指数与对照组差异无统计学意义。预防试验表明:mpt64-卡介苗重组疫苗及卡介苗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。结核分枝杆菌攻击后2个月,处死小鼠时,小鼠脏器大体病变、脏器培养、抗体检测结果及淋巴细胞增殖结果,各组间差别无统计学意义。初步实验结果表明:mpt64-卡介苗重组疫苗对结核分枝杆菌感染有明显的预防作用,但预防效果与卡介苗相比没有提高。

It was aimed to construct mpt64-recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively. The gene of MPT64 was amplified by PCR and recombined with shuttle plasmids pYUB295. The recombinant shuttle plasmid was identified by PCR, enzyme digestion and DNA sequencing 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 recombinant BCG vaccines was tested by their prevention and treatment experiments. PCR, enzyme digestion, DNA sequencing and SDS-PAGE results showed： The mpt64- recombinant BCG was constructed successfully and can express extrinsic MPT64 gene. The immunity experiment showed：extrinsic gene can be expressed in BCG and can stimulate B cell 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. The prevention experiment of recombinant BCG against M. tuberculosis was indicated： mpt64-recombinant BCG and BCG can extend mean time to death and reduce death rate in 2 month of those mouse, the protection effect of mpt6#- recombinant BCG had no difference with BCG. It could be concluded that the mpt64-ecombinant BCG was constructed successfully. The protection effect of mpt64-recombinant BCG had no difference with BCG.