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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
OBJECTIVE: To study the effect of plasmids expressing interleukin 18 gene on immune response induced by Mycobacterium tuberculosis (MTB) H37Rv Ag85A gene vaccine. 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 the eukaryotic expression vector pcDNA3.1 BamH Ⅰ and EcoR Ⅰ restriction sites. BALB / c mice were immunized intraperitoneally with the eukaryotic expression plasmid pcIL18 and MTBpcAg85A gene vaccine respectively expressing human IL-18 gene for 3 times at intervals of 2 weeks. Two weeks after each immunization, blood was collected and sera were separated. Titers of anti-Ag85A antibodies in sera of mice were measured by ELISA. Results: IL 18 cDNA was successfully amplified from RNA of human PMBCs by RT PCR and the sequencing result was correct. Identification by restriction enzyme digestion with BamH I and EcoR I confirmed that the gene of interest was inserted into the vector pcDNA3.1 and the positive clone was named pcIL18. In combination with pcIL18, the titer of serum anti-Ag85A antibody after three immunizations was significantly higher than that of pcAg85A alone. Conclusion: The combination of pcIL18 and pcAg85A gene vaccine can significantly enhance the humoral immune response of Ag85A antigen. Whether the combined immunization of pcIL18 + pcAg85A gene has the effect of enhancing Ag85A antigen-specific cellular immunity needs further study