【作者】 张灵霞；

【导师】 吴雪琼；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 免疫学， 2005， 博士

【摘要】 目的:构建结核分枝杆菌重组卡介苗,研究其免疫原性及抗结核作用,以期获得结核病预防性和治疗性疫苗。 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、MPT64、Ag85A、Ag85B的编码基因分别与穿梭质粒载体PMD31、pYUB295重组,通过电穿孔技术导入卡介苗中,应用PCR扩增、PAGE电泳鉴定重组卡介苗。将重组卡介苗免疫小鼠后,通过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;结核分枝杆菌攻击后,通过小鼠半数死亡时间、一定时间内的死亡率、肺、肝、脾细菌生长最低稀释度等指标评价各重组卡介苗对结核分枝杆菌感染的预防作用和治疗效果。 结果:通过PCR扩增获得esat6、mpt64、ag85a、ag85b基因,与穿梭表达载体PMD31、pYUB295重组后,通过PCR扩增、限制性内切酶酶切、DNA测序鉴定表明成功地构建了esat6、mpt64、ag85a、ag85b基因PMD31、pYUB295重组质粒。 将各重组质粒通过电穿孔导入卡介苗。质粒PMD31重组卡介苗在抗性培养上不能很好生长,质粒pYUB295重组卡介苗在抗性培养基上生长良好。pYUB295-目的基因重组卡介苗基因组DNA的PCR扩增以更多还原

【Abstract】 Objective: construct esat6 mpt6 ag85a ag85b recombinant BCG vaccine to find an improved vaccine to replace BCG and to prevent TB effectively.Methods: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR with primers designed according to the sequences publicized in Genebank and the vectors used. Those genes were recombined with shuttle plasmids pYUB295 and PMD31 respectively. The recombinant shuttle plasmids were identified by PCR enzyme digestion and DNA sequencing and then transformed into BCG by electroporation. The antibody was detected by ELISA and multiplication of mouse lymph-cell was detected by MTS.The mean time to death and death rate in 2 month et al were used to valued the protection of BCG recombinant vaccines.Results: The gene of esat6 mpt64 ag85a ag85b were amplified by PCR. pYUB295 and PMD31 recombinants of these genes were constructed through recombinant technology and identified them by PCR enzyme digestion and DNA sequencing. The recombinant plasmids were transformed into BCG respectively by electroporation. PMD31recombinant BCG could not grow very well in kanamycin resistance 7H10 culture medium, then, pYUB295 recombinant BCG was used for further study.The pYUB295 recombinant BCG were identified by amplified recombinant BCG genome DNA and showed pYUB295 recombinant Plasmids were transformed into BCG successfully. The SDS-PAGE results of pYUB295 recombinant BCG culture filtrate showed: the extrinsic esat6 mpt64 ag85a ag85b gene could be expressed in BCG.The weight of the mouse injected with recombinant BCG or BCG had no difference with the negative control and it indicated: recombinant BCG was safe and had no side effect. The antigen-specific antibody levels of mouse immunized with recombinant BCG were evaluation by ELISA using ESAT6 MPT6 Ag85A Ag85B protein and PPD as antigen. The ELISA results indicated: Each extrinsic gene can be express in BCG and can stimulate B cell of mouse to produce antibody; ag85a- recombinant BCG group had the highest antibody level, the antibody level had already improved after immunized 15 days and reached the peak after 45 days.The mouse lymph-cell stimulation test revealed: The lymph-cells of each group multiplicated when stimulated by different antigen such as conA ESAT6, MPT64 . Ag85A Ag85B protein and PPD respectively, all stimulation index reached 2. In other words, each protein could stimulate the mouse lymph-cell of each group obviously. The stimulationindex of each group had no obviously difference except esat6-recombiant BCG group.The mean time to death of ag85a^ ag85K esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse after injected with M.tuberculosis was 3K 29> >60^ 32> 30 and 13days respectively.The death rate in 2 month of those mouse was 60%, 50%, 30% ^ 50% > 60% and 80% respectively.The results of pathological changes and organ culture indicated: esat6-recombinant BCG group had less burden of M. tuberculosis than other groups. The antibody level showed: the antibody level of ag85a-recombinant BCG group was higher than other groups. The mouse lymph-cell stimulation test revealed: all stimulation indexes of different groups stimulated by different antigen were not obviously different.The mean time to death f ag85a^ ag85b> esat6> mpt64-recombianat BCG^ BCG and saline immuned mouse in the treatment experiment was 24 > 25> 3 K 23 -? 24 and 14days respectively.The death rate in 2 month of those mouse was 60% > 60%^ 50% > 50% > 60% and 70% respectively. Other experiment such as: the pathological changes, the organ culture, the antibody level and the mouse lymph-cell stimulation test were all consistent with their results in the prevention experiment.Conclusion: The esat6^ mpt64> ag85a> ag85b recombinant BCG were constructed successfully. BCG and recombinant BCG can prevent M. tuberculosis infection, but esat6-recombinant BCG was more ffective than更多还原