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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?
OBJECTIVE: To study the effect of plasmids expressing IL-12 and IL-18 gene on the immune response induced by CFP10 gene of Mycobacterium tuberculosis (MTB) strain H3 7Rv. METHODS: RNA was extracted from normal human peripheral blood mononuclear cells (PMBCs), IL 18 cDNA was amplified by RT-PCR and cloned into vector pGEM Teasy. After sequencing confirmed, subcloned into the eukaryotic expression vector pcDNA3.1 BamHI and EcoRI restriction sites. The eukaryotic expression plasmids pcmIL12 and pcIL18 which express mouse IL 12 and human IL 18 gene respectively were immunized with BALB / c mice by intramuscular injection with MTBCFP10 gene vaccine for 3 times at 2wk intervals. Serum was collected 2wk after each immunization and the serum was separated. The serum anti-CFP10 antibody titer was detected by ELISA. Results: IL 18 cDNA was successfully amplified from RNA of human PMBC by RT PCR and the sequencing result was correct. Identification by restriction enzyme digestion with BamHI and EcoRI confirmed that the gene of interest had been inserted into the vector pcDNA3.1 and the positive clone was named pcIL18. After the first immunization of the pc CFP10 group, the mean serum anti-CFP10 antibody titer was 1: 600 and the titer after the last immunization was 1: 400. After combined immunization with pcIL18 + pcCFP10, the titer of serum anti-CFP10 antibody was higher than that of pcCFP10 group, finally reaching 1: 800. However, the combined titers of pcmIL12 + pcCFP10 group were only 1: 200. Conclusion: The combination of pcIL18 and CFP10 gene vaccine can enhance the specific humoral immune response of CFP10 antigen, and pcmIL12 can reduce the antibody level of CFP10 gene vaccine. Does pcIL18 + pcCFP10 gene co-immunization have?