轮状病毒NSP4胞质区在杆状病毒系统的表达及其黏膜佐剂效应评价

Expression of rotavirus NSP4 cytoplasmic fragment in baculovirus system and its mucosal adjuvant effects

目的:应用Bac-to-Bac杆状病毒系统表达轮状病毒(RV)非结构蛋白NSP4的胞质区(CF)片段(47~175 aa),并初步检测其黏膜免疫佐剂效应。方法:将RV NSP4-CF基因片段定向克隆至转座载体pFastBac1,转化DH10Bac并筛选重组杆粒Bac-NSP4-CF。脂质体介导Bac-NSP4-CF转染sf9细胞,收获细胞并传代培养。测定病毒滴度,Western blot检测目的蛋白表达情况。纯化后的重组NSP4-CF与卵清蛋白(OVA)滴鼻共免疫小鼠,并检测其黏膜免疫佐剂效应。结果:在杆状病毒系统中成功获取了NSP4-CF重组蛋白,Western blot检测可见约16 kD的特异性条带;重组NSP4-CF与OVA共免疫小鼠后,小鼠血清OVA特异性IgG、IgA抗体水平与肠道SIgA水平较OVA单独免疫组均有升高(P<0.05);ELISPOT检测结果表明NSP4-CF佐剂组(5μg)分泌IFN-γ的脾细胞平均数均高于OVA组(P<0.05)。结论:经昆虫细胞表达系统可获得RV NSP4胞质区(47~175 aa)重组蛋白,该蛋白与模式抗原经黏膜途径免疫小鼠具有增强系统免疫应答与黏膜免疫应答的佐剂活性,为进一步研究RV-NSP4佐剂活性区域及其分子机制提供了实验基础。

Objective:To express cytoplasmic fragment(CF)of rotavirus(RV)NSP4 protein(47~175 aa)by applying Bac-to-Bac baculovirus vector system,and evaluate mucosal adjuvant effects of this recombinant protein.Methods:RV NSP4-CF gene fragment was directional cloned into shuttle vector pFastBac1,then pFastBac-NSP4-CF plasmids were transformed into E.coli DH10Bac cells.Recombinant bacmid Bac-NSP4-CF was selected to transfect sf9 insect cells via lipofectin reagent.Sf9 cells were harvested and passaged serially.The virus titers were determined,and recombinant NSP4-CF protein was analyzed by Western blot.Afterwards,mice were immunized by model antigens ovalbumin(OVA)with purified NSP4-CF so that the antigen-indued mucosal immune and adjuvant effects of NSP4-CF could be evaluated.Results:Recombinant protein of NSP4 cytoplasmic region was successfully expressed in baculovirus system,and a specific band of 16 kD was detected by Western blot.Levels of mouse serum OVA specific IgG,IgA antibody and intestinal SIgA immunized with recombinant NSP4-CF and OVA were significantly higher than those in OVA alone immunization group(P<0.05).Results of ELISPOT assay showed that average number of splenocytes secreting IFN-γin NSP4-CF adjuvant group(5μg)was higher than that in OVA group(P<0.05).Conclusion:Recombinant protein of RV NSP4-CF(47~175 aa)can be obtained by insect cell expression system.The protein has adjuvant properties to enhance systemic and mucosal immune responses via mucosal route in mice.This provides experimental basis on further research to locate its bioactivity region and clarify the adjuvant effects mechanism of the RV-NSP4..