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

Construction of DNA vaccine encoding of Ag85A gene of mycobacterium tuberculosis and observation on the immune responses in mice induced by co-immunization of Ag85A gene vaccine and plasmid encoding human interleukin 12

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。

Objective：To construct DNA vaccine based on Ag85A gene of mycobacterium tuberculosis,and to observe cellular immune responsees in mice induced by co-immunization of Ag85A gene vaccine and plasmid encoding human interleukin 12.Methods：（1）Constructing eukaryotic expressing plasmid.The gene encoding Ag85A mature protein was amplified by polymerase chain reaction（PCR）from genome of mycobacterium tuberculosis H37Rv strain,which inserted into cloning vector PMD20-T after restriction endonuclease digestion.The gene fragment encoding Ag85A mature protein was correctly inserted into the vector,confirmed by restriction endonuclease digestion and partial nucleotide sequencing,and then was subcloned to the corresponding sites of eukaryotic expressing vector pcDNA3.1（＋）.（2） Immunzation：50 C57BL/6N mice were randomly divided into following groups：① Ag85A gene vaccine plus plasmid of PORF-hlL-12;②Ag85A gene vaccine alone;③BCG（positive control）;④empty vector alone（negative control）;⑤ PBS（blank group）.The mice were immunized intramuscularly in both hind limbs 3 times at the intervals of three weeks or once subcutaneously with 1×106 of viable BCG（about 0.3 ml per mouse）at the first time of the DNA immunization.At the 28th days after the last immunization,the mice were killed,were splenocytes isolated.The splenocytes cytotoxicity-mediated was detected by LDH release assay,and proliferation of splenocyte was detected by XTT after stimulation of TB-PPD.The level of IFN-γ,IL-2 and IL-4 in the supernatant of spleno-lymphocyte cultures was measured by EILSA.Results：（1）The DNA vaccine entcoding Ag85A gene of mycobacterium tuberculosis was successfully constructed.（2）The co-immunization group could induce stronger Th1 cellular immune response.The levels of IFN-γ and IL-2 in supernatant of spleno-lymphocyte cultures in the co-immunization group were significantly higher than that of group of Ag85A alone,and they were similar to that in BCG group.The level of IL-4 decreased and was lower than that of BCG group.Specific spleen T cell cytotoxicity and splenocyte proliferation activities were significantly stronger than the other groups.Conclusion：Eukaryotic expressing plasmid PORF-hIL-12 can significantly reinforce the specific cellular immune responses in mice induced by the Ag85A DNA vaccine.