寨卡病毒包膜蛋白重组人腺病毒5型载体的构建与免疫原性测定

目的 以腺病毒5型载体（Ad5）构建含寨卡病毒（ZIKV）包膜蛋白（prM-E）的复制缺陷型5型重组腺病毒（rAd5）表达载体，测定prM-E在细胞中的表达及对小鼠的免疫原性。方法 从ZIKV毒株Z16006（亚洲型）分离获得prM-E基因片段，以重组腺病毒AdMax^TM系统包装重组腺病毒rAd5/prM-E。选用C57BL/6小鼠，以rAd5/prM-E 10⁷、10⁸和10⁹ PFU三个剂量肌内注射免疫小鼠，在第3周同等剂量加强免疫一次，第5周从小鼠眼球取血并分离脾淋巴细胞。分别以ELISpot和ELISA方法测定小鼠对ZIKV prM-E的体液与细胞免疫反应。结果 成功构建复制缺陷型重组腺病毒载体rAd5/prM-E，采用Western blot方法和抗ZIKV E抗体检测rAd5/prM-E感染的293A细胞表达产物，可见与E蛋白相应的56 kDa蛋白带。以ELISpot方法测定小鼠脾淋巴细胞特异性IFN-γ分泌细胞形成斑点数（SFCs），结果分别为（688.54±186.43）、（1 084.90±144.14）和（1 640.20±147.13） SFCs/10⁶脾细胞，与病毒接种剂量呈正比关系；采用ELISA测定免疫小鼠血清中抗E抗体，其滴度（log10值）分别为（3.14±0.39）、（3.50±0.30）和（3.74±0.25）， 均高于对照组小鼠（0.80±0.17），差异具有统计学意义（P<0.001）。结论 rAd5/prM-E具有感染小鼠并诱导小鼠产生强烈的特异性抗体和细胞免疫反应的能力，表明prM-E具有良好的免疫原性，为ZIKV候选疫苗研制提供了可靠的免疫源。

Construction and immunogenicity detection of recombinant human adenovirus type 5 vector containing ZIKV envelope protein

Objective To construct replication-defective recombinant adenovirus type 5 (rAd5) vector containing Zika virus (ZIKV) envelope protein prM-E, and determine the expression of prM-E in cells and its immunogenicity in mice. Methods prM-E gene fragment was obtained from ZIKV strain Z16006 (Asian type),recombinant adenovirus rAd5/prM-E was enveloped with recombinant adenovirus AdMax^TM system. C57BL/6 mice were immunized by intramuscular injection of rAd5/prM-E at three doses(10⁷, 10⁸ and 10⁹ PFU), mice were immunized again at the same dose at week 3, blood was taken from the eyeballs of mice and spleen lymphocytes were separated at week 5. Humoral and cellular immune response of mice to ZIKV prM-E were detected by ELISpot and ELISA respectively. Results The replication-defective recombinant adenovirus vector rAd5/prM-E was constructed successfully, expression products of 293A cells infected with rAd5/prM-E were detected by Western blot and anti-ZIKV E antibody, 56 kDa protein band which corresponded with E protein was observed. Spots forming cells (SFCs) secreted by mice splenic lymphocyte specific IFN-γ was detected by ELISpot method, results were (688.54±186.43), (1 084.90±144.14), and (1 640.20±147.13) SFCs/10⁶ splenic cell respectively, which were positively proportional to the immune dose; anti-E antibody in serum of immunized mice was determined by enzyme-linked immunosorbent assay (ELISA), and the titers (log10 value) were (3.14±0.39), (3.50±0.30), and (3.74±0.25) respectively, all were significantly higher than(0.80±0.17) of control group (P<0.001). Conclusion rAd5/prM-E has the ability to infect mice and induce strong specific antibodies and cellular immune response, which indicates that prM-E has good immunogenicity and provides a reliable immune source for the development of ZIKV candidate vaccine.