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

发布时间：2019-03-19 14:09

【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
[Abstract]:Aim: to construct recombinant BCG vaccine of Mycobacterium tuberculosis and study its immunogenicity and anti-tuberculosis effect in order to obtain preventive and therapeutic vaccine against tuberculosis. Methods: the gene encoding the protective antigen ESAT6,MPT64,Ag85A,Ag85B of Mycobacterium tuberculosis was recombined with shuttle plasmid vector PMD31,pYUB295 by gene engineering technique. The gene was introduced into BCG by electroporation technique and amplified by PCR. Recombinant BCG was identified by PAGE electrophoresis. After the mice were immunized with recombinant BCG vaccine, the specific antibodies in serum were detected by ELISA method, and the proliferation index of spleen lymphocytes was analyzed by MTT method. After Mycobacterium tuberculosis infection, the preventive and therapeutic effects of recombinant BCG vaccine on Mycobacterium tuberculosis infection were evaluated by the indexes of half death time, mortality within a certain time, minimum dilution of lung, liver and spleen bacteria growth and so on. Results: esat6,mpt64,ag85a,ag85b gene was amplified by PCR. After recombination with shuttle expression vector PMD31,pYUB295, esat6,mpt64,ag85a,ag85b gene PMD31, was successfully constructed by PCR amplification, restriction endonuclease digestion and DNA sequencing. PYUB295 recombinant plasmid. The recombinant plasmids were introduced into BCG vaccine by electroporation. Plasmid PMD31 recombinant BCG did not grow well in resistant culture and plasmid pYUB295 recombinant BCG grew well on the resistant medium. PCR amplification of genomic DNA of pYUB295-target recombinant BCG
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

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