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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。
Recombinant plasmids were introduced into BCG by electroporation and amplified by polymerase chain reaction (PCR). Polyacrylamide (PMMA) Gel electrophoresis (PAGE) Identification of mpt64-BCG recombinant vaccine: MPT64 gene pYUB295 recombinant plasmid was successfully constructed and MPT64 protein was secreted and expressed in BCG. BCG recombinant vaccine immunogenicity tests showed that: only mpt64-BCG recombinant vaccine group MPT64-specific antibodies, 45 days to reach the highest level. Splenic lymphocyte proliferation test showed that the average stimulation index of mice in each group reached more than 2.0. There was no significant difference in stimulating index of splenic lymphocytes between mpt64-BCG recombinant vaccine group and control group. Prophylaxis test showed that both mpt64-BCG vaccine and BCG-B can prolong the half-life of Mycobacterium tuberculosis infected mice and reduce the mortality within 2 months. Mycobacterium tuberculosis 2 months after the mice were sacrificed, the general pathological changes in organs of mice, organ culture, antibody test results and lymphocyte proliferation results, the difference between the groups was not statistically significant. Preliminary experimental results show that: mpt64 - BCG vaccine recombinant Mycobacterium tuberculosis infection has a significant preventive effect, but the preventive effect compared with BCG did not increase.