细胞因子IL-18基因对Ag85A DNA疫苗诱导产生抗体水平的影响

目的 :研究表达含白介素 (IL) 18基因的质粒 ,对结核分枝杆菌 (MTB)H37Rv Ag85A基因疫苗诱导免疫应答的影响。方法 :从正常人外周血单核细胞 (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 ,亚克隆真核表达载体pcDNA3.1的BamHⅠ和EcoRⅠ酶切位点。将分别表达人IL 18基因的真核表达质粒pcIL18与MTBpcAg85A基因疫苗联合肌注免疫BALB c小鼠 ,共免疫 3次 ,每次间隔 2周。每次免疫后 2周采血 ,分离血清 ,用ELISA检测小鼠血清抗Ag85A抗体的滴度。结果 :用RT PCR成功地从人PMBCs的RNA中扩增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。联合应用pcIL18,三次免疫后血清抗Ag85A抗体的滴度明显较单独应用pcAg85A增高。结论 :pcIL18与pcAg85A基因疫苗联合免疫 ,可显著增强Ag85A抗原的特异性体液免疫应答。pcIL18+pcAg85A基因联合免疫是否具有增强Ag85A抗原特异性细胞免疫的作用有待进一步研究

Effects of Cytokine IL-18 Gene on Antibody Production Induced by Ag85A DNA Vaccine

Objective: To investigate the effects of plasmid containing human IL-18 gene on the humoral immune response of mice immunized by Ag85A DNA vaccines of Mycobacterium tuberculosis H37 Rv strain. Methods:Human IL-18 cDNA was amplified from RNA of peripheral blood mononuclear cells(PBMCs )by RT- PCR and cloned into the pGEM-TEasy vector. After sequencing IL-18 gene was subcloned into the the sites of Bam H I and EcoR Ⅰ digestion of pcDNA3.1. BALB/c mice were injected intramuscularly with eukaryotic expression plasmid pcIL18, together with MTB pcAg85A DNA vaccines. The same immunization was repeated three times at intervals of two weeks. Mouse sera were collected at two weeks after the each injection. The titers of anti-Ag85A antibody were 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 H-18 gene was correctly inserted into the vector pcDNA3.1, which was confirmed with BamH I and EcoR I digestion analysis. The positive plasmid was called pcIL18.After being immunized with DNA vaccines,the titers of antibody obtained from mice being immunized by pcAg85A combining with pcIL18 were superior to mice immunanized by pcAg85A independently. Conclusion: Combination of IL-18 gene with MTB pcAg85A DNA vaccine could observably enhance the humoral immune responses to pcAg85A. It remains further investigated whether IL-18 gene plus MTB pcAg85A DNA vaccine could markedly induce the cellular mediated immune response to Ag85A or not.