融合蛋白RAP的免疫原性分析

Immunogenicity Analysis of the Fusion Protein RAP

目的结核病(Tuberculosis,TB)目前是全球单一感染源造成死亡的主要原因之一,且广泛接种的卡介苗对于不同人的效果不一且有一定的副作用,甚至在某些成年人身上效果甚微,为了获得效果更好、安全性更好的结核疫苗对融合蛋白RAP进行了分析验证。方法将融合蛋白RAP连接到pET30b原核表达载体上通过双酶切鉴定构建重组质粒、用热休克法和诱导剂诱导蛋白表并通过Ni-NTA纯化蛋白;全血干扰素释放测定法(WBIA)检测RAP刺激的人外周血中IFN-γ的水平;ELISA法检测小鼠特异性IgG、IgG1和IgG2a以及IFN-γ、TNF-α、IL-2和IL-4的分泌水平。结果成功构建pET30b-RAP重组质粒并表达纯化RAP蛋白;RAP及其各组成蛋白能明显导致国内M.tb感染者分泌高水平的IFN-γ;小鼠经过RAP/MTO免疫后会发生Th1型反应产生特异性高滴度IgG抗体且使抗原特异性Th1型细胞因子分泌能力明显增强。结论根据得到的实验结果推断出RAP具有强大且特异性的免疫能力,所以有希望成为一种有前景的结核病候选疫苗。

Objective To obtain a tuberculosis vaccine with a better efficacy and better safety with the fusion protein RAP being analyzed and validated since tuberculosis(TB)is currently one of the leading causes of death from a single source of infection worldwide and the widely vaccinated BCG vaccine has varying effects and side effects in different people,even little effect in some adults.Methods The fusion protein RAP was ligated into the pET30b prokaryotic expression vector and the recombinant plasmid was constructed by double enzyme digestion.The protein sheet was induced by the heat shock method and inducer,and the protein was purified by Ni-NTA.The level of IFN-γin RAP-stimulated human peripheral blood was detected by whole-blood interferon-releasing assay(WBIA)and the specific IgG,IgG1 and IgG2a,as well as the secretion levels of IFN-γ,TNF-α,IL-2 and IL-4,in mice were detected by ELISA.Results The pET30b-RAP recombinant plasmid was successfully constructed and the RAP protein was expressed and purified.RAP and its constituent proteins significantly led to the secretion of high levels of IFN-γin M.tb-infected mice.The mice immunized with RAP/MTO underwent a Th1-type reaction to produce specific high-titer IgG antibodies and the secretion of antigen-specific Th1-type cytokines was significantly enhanced.Conclusion Based on the experimental results obtained,it is inferred that RAP has a strong and specific immunizing ability,and therefore has the potential to be a promising vaccine candidate for tuberculosis.