表达诺如病毒衣壳蛋白的重组腺病毒疫苗的构建及其免疫原性分析

Construction and immunogenicity of recombinant adenovirus vaccine expressing capsid protein of norovirus

目的 构建表达诺如病毒(norovirus,NoV)衣壳蛋白VP1和VP2基因的重组腺病毒疫苗,并检测其在小鼠体内的免疫原性。方法 以5型腺病毒为载体,制备表达NoV主要衣壳蛋白VP1以及同时表达VP1、VP2和3′UTR区的两种重组腺病毒载体,通过PCR和Western blot法对重组腺病毒进行体外鉴定。将两组重组腺病毒以108IU/只的剂量灌胃免疫BALB/c小鼠,共免疫2次,间隔3周。采用ELISA和ELISPOT等方法检测两组重组腺病毒诱导的体液免疫、黏膜免疫和细胞免疫反应。结果 重组腺病毒pkAd5ES-VP1和pkAd5ES-VP1-2的病毒滴度分别为3.4×10;和2.4×10;IU/mL。两种重组腺病毒均能在真核细胞中高效表达外源基因。VP1+VP2+3′UTR区共表达的重组腺病毒在灌胃免疫时能有效提升小鼠粪便IgA、血清组织血型抗原(histo-blood group antigen,HBGA)阻断抗体水平以及脾脏IL-4分泌细胞的数量。结论 与单独表达VP1比较,VP1+VP2+3′UTR区共同表达的重组腺病毒疫苗具有更好的免疫原性。本研究为高效口服NoV疫苗的研发提供了参考。

Objective To construct recombinant adenovirus vaccine expressing capsid proteins VP1 and VP2 of capsid protein of norovirus(NoV) and determine its immunogenicity in mice. Methods Two recombinant adenovirus plasmids were prepared using adenovirus type 5 as a vector,of which one contained the gene encoding major capsid protein VP1while the other contained those encoding VP1 and VP2 proteins and 3′ UTR of NoV. The recombinant adenovirus was identified in vitro by PCR and Western blot. BALB/c mice were immunized with the two recombinant adenovirus vaccines each at a dosage of 108IU by gavage for 2 times at an interval of 3 weeks. The induced humoral,mucosal and cellular immune responses were determined by ELISA and ELISPOT. Results The titers of recombinant adenoviruses pkAd5ES-VP1 and pkAd5ES-VP1-2 were 3. 4 × 10~9and 2. 4 × 10~9IU/mL respectively. Foreign genes were successfully expressed in eukaryotes by using both the recombinant adenoviruses. The recombinant adenovirus in which VP1 + VP2 +3′ UTR were co-expressed effectively increased the levels of fecal IgA and serum histo-blood group antigen(HBGA)blocking antibody as well as the number of IL-4 secreting cells in spleen of mice immunized by gavage. Conclusion Compared with that for expression of VP1 alone,the recombinant adenovirus vaccine for co-expression of VP1 + VP2 +3′ UTR showed high immunogenicity. This study provided a reference for the development of an effective oral norovirus vaccine.