结核分枝杆菌Ag85A基因DNA疫苗的构建及其联合人IL-12表达质粒诱导的小鼠细胞免疫应答观察

发布时间：2018-05-28 23:20

本文选题：结核分枝杆菌 + AgA基因　；参考：《中国免疫学杂志》2011年01期

【摘要】：目的:构建编码结核分枝杆菌Ag85A分泌蛋白重组真核表达质粒,研究其与hIL-12联合免疫小鼠后的细胞免疫应答。方法:(1)构建质粒:采用PCR法从H37Rv菌株中扩增Ag85A编码基因,用限制性内切酶消化后,插入克隆载体PMD20-T中,经酶切鉴定与序列测定证实后,以亚克隆法构建于真核表达载体PCDNA3.1的相应酶切位点。(2)动物实验:50只C57BL/6N小鼠随机分为:①Ag85A基因疫苗+hIL-12质粒组(联合免疫组);②重组Ag85A基因疫苗组;③卡介苗BCG组(阳性对照);④空载体组(阴性对照);⑤PBS组(空白对照)。基因疫苗、空载体和PBS经肌内注射法免疫各组小鼠,每隔3周免疫1次,共免疫3次,BCG组经尾部皮下注射1×106CFU BCG免疫1次,约0.3 ml/只。第三次免疫小鼠后28天,处死各组小鼠,分离脾细胞,ELISA法检测脾细胞培养上清液中IFNγ-、IL-2、IL-4水平;乳酸脱氢酶释放法检测脾细胞杀伤活性;分离的脾细胞经TB-PPD刺激后,XTT比色法检测脾淋巴细胞增殖活性。结果:(1)成功构建结核分枝杆菌Ag85A基因DNA疫苗。(2)联合免疫组能诱导较强烈的抗原特异性Th1型细胞免疫应答,免疫小鼠脾细胞培养上清液IFN-r和IL-2水平显著高于Ag85A基因疫苗组,与BCG组相当,IL-4分泌减少;特异性CTL杀伤活性明显增强;淋巴细胞增殖活性也明显高于其他组别。结论:hlL-12表达质粒能够增强结核分枝杆菌Ag85A基因DNA疫苗所诱导的小鼠免疫应答。
[Abstract]:Aim: to construct a recombinant eukaryotic expression plasmid encoding mycobacterium tuberculosis Ag85A secretory protein and study the cellular immune response of mice immunized with hIL-12. Methods: the plasmid was constructed: the Ag85A coding gene was amplified by PCR from H37Rv strain, digested with restriction endonuclease, inserted into clone vector PMD20-T, and confirmed by restriction endonuclease digestion and sequencing. In animal experiment: 50 C57BL/6N mice were randomly divided into 1 Ag85A gene vaccine hIL-12 plasmid group (combined immunized group) and recombinant Ag85A gene vaccine group. 3 BCG BCG group. The mice in each group were immunized with gene vaccine, empty vector and PBS intramuscularly once every 3 weeks. The mice in the control group were immunized with 1 脳 106CFU BCG subcutaneously once every 3 weeks, about 0.3 ml/. On the 28th day after the third immunization, the mice in each group were killed, the spleen cells were isolated and the levels of IL-4 in the supernatant of spleen cell culture were detected by Elisa, and the killing activity of splenocytes was detected by lactate dehydrogenase release method. The proliferative activity of splenic lymphocytes was detected by TB-PPD colorimetric assay. Results the Ag85A gene DNA vaccine of Mycobacterium tuberculosis was successfully constructed. The combined immunized group could induce a strong antigen-specific Th1 type cellular immune response. The levels of IFN-r and IL-2 in the supernatant of spleen cell culture of immunized mice were significantly higher than those in the Ag85A gene vaccine group. Compared with BCG group, IL-4 secretion was decreased, specific CTL cytotoxicity was increased, and lymphocyte proliferation activity was significantly higher than that in other groups. Conclusion the expression plasmid of 1: hlL-12 can enhance the immune response of mice induced by Ag85A gene DNA vaccine of Mycobacterium tuberculosis.
【作者单位】：重庆医科大学第二附属医院呼吸内科;
【基金】：国家自然科学基金资助项目(No.30872261)
【分类号】：R392.1

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