目的：研究LIGHT作为免疫佐剂在EB病毒核抗原1（EBNA1）DNA疫苗诱导免疫反应中的作用。方法以Balb/c小鼠肝脏cDNA为模板，PCR扩增构建真核表达载体pcDNA3.1-LIGHT；转染细胞，验证LIGHT蛋白的表达；采用电脉冲方法，以LIGHT质粒为佐剂与EBV DNA疫苗联合免疫小鼠，测定血清中EBNA1特异性抗体；分离和收集小鼠脾脏淋巴细胞，经EBNA1抗原刺激后，测定细胞因子干扰素-γ（IFN-γ）的分泌水平。结果 PCR扩增出LIGHT目的片段，大小为720 bp；蛋白质免疫印迹法（Western blotting）鉴定结果：在蛋白Marker相对分子质量26×103上方有单一清晰目的条带，表明有LIGHT蛋白表达；免疫小鼠血清测定实验结果：LIGHT联合DNA疫苗实验组的抗体滴度是单独DNA疫苗免疫组的3倍；脾淋巴细胞因子测定结果表明LIGHT联合免疫组能明显增强IFN-γ的分泌。结论 LIGHT作为免疫佐剂，可依赖其介导的信号通路对抗体的产生和相关细胞因子的分泌具有显著促进作用。%Objective To study the effect of LIGHT as an adjuvant in the immune responses induced by EB viral nuclear antigen1(EBNA1) DNA vaccine. Methods The liver cDNA of Balbc/c mouse served as the template. The eukaryotic expression vector pcDDNA3.1-LIGHT was constructed by PCR amplification;the cell transfection was conducted,the LIGHT protein expres-sion was verified;by adopting the electric pulse method,the mouse was immunized with the LIGHT plasmid as the adjuvant com-bined with EBV DNA vaccine. The specific antibody of serum EBNA1 was detected. The mouse spleen lymphocytes were separat-ed and collected. After stimulation by EBNA1 antigen ,cytokine IFN-γsecretion level was detected. Results The LIGHT target fragment was amplified by PCR,which was about 720 bp,the Western blot identification results showed that a single clear target band was above 26 ×103,demonstrating that LIGHT protein was expressed;the serum detection results in the immune mouse showed that the antibody titer in the LIGHT combined DNA vaccine experimental group was 3 times of that in the single DNA vac-cine immune group;the spleen lymphocyte factor detection results showed that the LIGHT combined immune group could signifi-cantly increase the IFN-γsecretion. Conclusion LIGHT as the adjuvant has significant promoting effect in the generation of an-tibody and secretion of related cytokines by depending on its mediated signal pathway.
展开▼
