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目的构建串联表达口蹄疫病毒VP1-2A和猪干扰素-α的重组腺病毒,并研究重组病毒对小鼠特异性免疫的影响。方法将FMDV VP1-2A基因和α干扰素基因先后克隆到腺病毒的穿梭载体中,得到的阳性穿梭质粒与腺病毒骨架载体在大肠杆菌BJ5183中进行同源重组。重组腺病毒质粒感染HEK293A细胞,获得重组腺病毒rAd5VP1-2A-poIFN-α。用rAd5VP1-2ApoIFN-α免疫BALB/c小鼠,同时设注射单独表达FMDV VP1的重组腺病毒(rAd5VP1)和空腺病毒的对照组。通过ELISA检测血清中特异性IgG、IgG1、IgG2a及细胞因子IL-4、IFN-γ的表达水平;MTT法检测淋巴细胞增殖指数。结果成功构建了共表达FMDV VP-2A和IFN-α的重组腺病毒rAd5VP1-2A-poIFN-α。小鼠免疫实验结果显示,与单独表达FMDV VP1基因的重组腺病毒rAd5VP1相比,重组腺病毒rAd5VP1-2A-poIFN-α能增强特异性IgG、IgG1和IgG2a的分泌,促进IL-4和IFN-γ的分泌,并能增强小鼠淋巴细胞增殖功能。结论重组腺病毒rAd5VP1-2A-poIFN-α能明显增强小鼠特异性免疫应答。
Objective To construct a recombinant adenovirus expressing VP1-2A and porcine interferon-α in tandem and study the effect of the recombinant virus on the specific immunity in mice. Methods The FMDV VP1-2A gene and interferon alpha gene were cloned into the shuttle vector of adenovirus. The positive shuttle plasmid was homologously recombined with adenovirus backbone vector in E. coli BJ5183. Recombinant adenovirus plasmid was infected HEK293A cells to obtain recombinant adenovirus rAd5VP1-2A-poIFN-α. BALB / c mice were immunized with rAd5VP1-2ApoIFN-α and injected with a recombinant adenovirus (rAd5VP1) expressing FMDV VP1 alone and a control group with empty adenovirus. Serum levels of specific IgG, IgG1, IgG2a and cytokines IL-4 and IFN-γ were detected by ELISA. The proliferation index of lymphocytes was detected by MTT assay. Results The recombinant adenovirus rAd5VP1-2A-poIFN-α co-expressing FMDV VP-2A and IFN-α was successfully constructed. The results of mouse immunization showed that the recombinant adenovirus rAd5VP1-2A-poIFN-α enhanced the secretion of specific IgG, IgG1 and IgG2a, and promoted the expression of IL-4 and IFN-γ. Compared with recombinant adenovirus rAd5VP1 expressing FMDV VP1 gene alone, γ secretion, and can enhance mouse lymphocyte proliferation. Conclusion The recombinant adenovirus rAd5VP1-2A-poIFN-α can significantly enhance the specific immune response in mice.