Th1型细胞因子基因对结核分枝杆菌基因疫苗诱导BALB/c小鼠产生抗CFP10抗体水平的影响

Effects of Th1 cytokine gene on anti-CFP10 antibody production in BALB/c mice induced by Mycobacterium tuberculosis DNA vaccine

目的 :研究分别表达含IL 12和IL 18基因的质粒 ,对结核分枝杆菌 (Mycobacteriumtuberculosis,MTB)H3 7Rv 株CFP10基因疫苗诱导免疫应答的影响。方法 :从正常人外周血单个核细胞 (PMBCs)中提取RNA ,用RT PCR扩增IL 18cDNA ,并克隆入载体 pGEM Teasy中。测序证实后 ,亚克隆至真核表达载体 pcDNA3.1的BamHI和EcoRI酶切位点。将分别表达小鼠IL 12和人IL 18基因的真核表达质粒pcmIL12和pcIL18,与MTBCFP10基因疫苗联合肌注免疫BALB/c小鼠 ,共免疫 3次 ,每次间隔 2wk。每次免疫后 2wk采血、分离血清 ,用ELISA检测小鼠血清抗CFP10抗体的滴度。结果 :用RT PCR成功地从人PMBC的RNA中扩增出IL 18cDNA ,测序结果正确。用BamHI和EcoRI酶切鉴定证实 ,目的基因已插入载体 pcDNA3.1中 ,阳性克隆命名为pcIL18。pc CFP10组第 1次免疫后 ,血清抗CFP10抗体的平均滴度为 1∶6 0 0 ,末次免疫后的滴度为 1∶4 0 0 0。pcIL18+pcCFP10组联合免疫后 ,血清抗CFP10抗体的滴度高于 pcCFP10组 ,最终达 1∶80 0 0。而pcmIL12 + pcCFP10组联合免疫后滴度仅为 1∶2 0 0。结论 :pcIL18与CFP10基因疫苗联合免疫 ,可增强CFP10抗原的特异性体液免疫应答 ;pcmIL12则可使CFP10基因疫苗产生的抗体水平降低。pcIL18+ pcCFP10基因联合免疫是否具有?

AIM: To investigate the effects of plasmid containing mouse IL 12 and human IL 18 genes on the humoral immune response of mice immunized by CFP10 gene of Mycobacterium tuberculosis (MTB) H 37 R v strain. METHODS: Human IL 18 cDNA was amplified from RNA of PBMCs by RT PCR and cloned into the pGEM Teasy vector. After sequencing it was subcloned into the the sites of Bam H I and Eco R I digestion of pcDNA3.1. BALB/c mice were injected intramuscularly by eukaryotic expression plasmid pcmIL12 and pcIL18, together with MTB CFP10 DNA vaccine, respectively. The same immunization repeated three times at intervals of two weeks. Mouse sera were collected at two weeks after the each injection. The titer of anti CFP10 antibody was detected by ELISA. RESULTS: IL 18 cDNA was amplified successfully from RNA of human PBMCs by RT PCR and the result of sequencing was correct. The IL 18 gene was correctly inserted into the vector pcDNA3.1 by being confirmed with Bam H I and Eco R I digestion analysis, positive plasmid was called pcIL18. After being immunized with pcCFP10 three times, the end point titer of anti CFP10 was 1∶4 000, while the titer obtained by being immunized with pcIL18+ pcCFP10 was 1∶8 000, but yet, after being immunized with pcmIL12+pcCFP10, the end point titer of anti CFP10 antibody was only 1∶200. CONCLUSION: Combination of IL 18 gene with MTB CFP10 DNA vaccine can enhance the humoral immune responses to pcCFP10, whereas the immunization with IL 12 gene plus pcCFP10 made humoral immune response markedly descent. As for whether IL 18 gene plus MTB CFP10 DNA vaccine can induce markedly the cellular mediated immune response to CFP10 or not remains to be further investigated.