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目的构建表达猪白细胞介素-2(poIL-2)的重组腺病毒,研究其作为佐剂对口蹄疫重组腺病毒免疫效果的影响。方法将poIL-2基因克隆到腺病毒的穿梭载体中,与腺病毒骨架载体共转化BJ5183感受态细胞,获得的重组病毒质粒通过脂质体转染HEK-293A细胞,获得表达poIL-2的重组腺病毒(rAd5poIL-2)。以rAd5poIL-2作为佐剂与表达PMDV VP基因的重组腺病毒(rAd5VP1)联合免疫小鼠,以单独免疫rAd5VP1的小鼠作为对照。通过测定淋巴细胞增殖指数、特异性抗体、IL-4和IFN-γ的水平来衡量rAd5poIL-2对rAd5VP1的免疫增强效果。结果经PCR鉴定,成功构建了rAd5poIL-2。MTT与抗体检测结果显示,rAd5poIL-2既能增强rAd5VP1诱导小鼠淋巴细胞增殖,又能促进rAd5VP1诱导小鼠特异性抗体的产生,细胞因子检测结果显示,rAd5poIL-2能促进rAd5VP1诱导Th1和Th2的分化。结论 rAd5poIL-2可望成为口蹄疫疫苗的候选佐剂。
Objective To construct a recombinant adenovirus expressing porcine interleukin-2 (poIL-2) and study its effect as an adjuvant on the immunogenicity of recombinant adenovirus of foot-and-mouth disease. Methods The poIL-2 gene was cloned into the shuttle vector of adenovirus and co-transformed with adenovirus backbone vector into competent BJ5183 cells. The recombinant plasmid was transfected into HEK-293A cells by lipofectamine to obtain recombinant poIL-2 Adenovirus (rAd5poIL-2). Mice were immunized with rAd5poIL-2 as adjuvant in combination with the recombinant adenovirus expressing the VPD gene of PMDV (rAd5VP1), and mice immunized with rAd5VP1 alone were used as controls. The effect of rAd5poIL-2 on the immune enhancement of rAd5VP1 was measured by measuring the level of lymphocyte proliferation index, specific antibodies, IL-4 and IFN-γ. Results After identification by PCR, rAd5poIL-2 was successfully constructed. The results of MTT and antibody showed that rAd5poIL-2 could enhance the proliferation of mouse lymphocytes induced by rAd5VP1 and promote the production of mouse specific antibodies by rAd5VP1. The results of cytokines showed that rAd5poIL-2 could promote the expression of Th1 and Th2 Differentiation. Conclusion rAd5poIL-2 is expected to be a candidate adjuvant for foot-and-mouth disease vaccine.