甲型肝炎病毒衣壳蛋白VP1、VP3的原核表达及其免疫原性  

Prokaryotic expression and immunogenicity of hepatitis A virus capsid proteins VP1 and VP3

作　　者：赵丹莹 周永飞 徐艳玲 陈子杨 唐剑光 常东英 王艺博 关诗宇 常军亮 曹玉锋 ZHAO Danying;ZHOU Yongfei;XU Yanling;CHEN Ziyang;TANG Jianguang;CHANG Dongying;WANG Yibo;GUAN Shiyu;CHANG Junliang;CAO Yufeng(Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130012

出　　处：《中国生物制品学杂志》2023年第5期524-530,共7页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(20190404001YY)。

摘　　要：目的原核表达甲型肝炎病毒(hepatitis A virus,HAV)衣壳蛋白VP1和VP3,并评价其免疫原性。方法PCR法扩增VP1和VP3基因片段,克隆至载体pET-G28a中,构建重组表达质粒pET-G28a-VP1和pET-G28a-VP3,转化至感受态E.coli BL21(DE3),经IPTG诱导表达,12%SDS-PAGE分析表达产物。目的蛋白经离子交换层析纯化,复性后与多种佐剂配伍,免疫雄性BALB/c小鼠,随机分为VP1-MF59、VP1-AH、VP1-AP、VP1-AP-10CpG、VP1-AP-50CpG、VP3-AP、VP3-VP1-AP及PBS对照组,每组5只。ELISA法检测各组小鼠血清中IgG抗体效价,快速荧光灶免疫抑制试验测定VP1-AP、VP3-VP1-AP及PBS对照组小鼠血清中和抗体效价。结果菌落PCR及测序结果证明,重组表达质粒pETG28a-VP1及pET-G28a-VP3构建正确。重组蛋白VP1和VP3相对分子质量分别约为37000和26000,主要以包涵体形式存在,表达量分别为18.6%和32.4%,纯度分别为86.3%和84.7%,且分别可与兔抗HAV抗血清及鼠抗HAVVP3抗血清发生特异性结合。VP1-AP-50CpG组小鼠血清的IgG抗体效价显著高于VP1-AP组(q=22.05,P<0.01),VP3-VP1-AP组小鼠血清的IgG抗体效价显著高于VP1-AP及VP3-AP组(q分别为22.05和22.49,P均<0.01)。与PBS对照组比较,VP1-AP及VP3-VP1-AP组小鼠血清的中和抗体效价显著升高(q分别为7.79和25.11,P<0.01)。结论原核表达的HAV衣壳蛋白VP1和VP3纯度较高,且具有良好的免疫原性,制剂中添加CpG有利于增强抗原的免疫原性。本研究为HAV重组亚单位疫苗的研发奠定了基础。Objective To express the capsid proteins VP1 and VP3 of hepatitis A virus(HAV)in prokaryotic cells and evaluate their immunogenicity.Methods VP1 and VP3 gene fragments were amplified by PCR,cloned into vector pETG28a to construct recombinant expression plasmids pET-G28a-VP1 and pET-G28a-VP3,which were transformed into competent E.coli BL21(DE3),induced by IPTG,and then analyzed by 12%SDS-PAGE.The target protein was purified by ion exchange chromatography,renatured and combined with several adjuvants to immunize mice.The mice were divided into VP1-MF59,VP1-AH,VP1-AP,VP1-AP-10CpG,VP1-AP-50CpG,VP3-AP,VP3-VP1-AP and PBS control groups,five for each group.Serum IgG antibody titers of mice in various groups were detected by ELISA,and serum neutralizing antibody titers of mice in VP1-AP,VP3-VP1-AP and PBS control groups were detected by rapid fluorescence focus immunosuppression experiment.Results Colony PCR and sequencing showed that the recombinant plasmids pET-G28a-VP1 and pET-G28a-VP3were constructed correctly.The recombinant proteins VP1 and VP3,with relative molecular masses of about 37000 and26000 respectively,mainly existed in the form of inclusion bodies,the expression levels were 18.6%and 32.4%,and the purity was 86.3%and 84.7%,respectively.The recombinant proteins VP1 and VP3 reacted specifically with rabbit anti-HAV antiserum and mouse anti-HAV-VP3 antiserum respectively.The serum IgG antibody titer of mice in VP1-AP-50CpG group was significantly higher than that in VP1-AP group(q=22.05,P<0.01),and the serum IgG antibody titer of mice in VP3-VP1-AP group was significantly higher than that in VP1-AP group and VP3-AP group(q=22.05 and 22.49 respectively,each P<0.01).Compared with PBS control group,the serum neutralizing antibody titer of mice in VP1-AP and VP3-VP1-AP group increased significantly(q=7.79 and 25.11 respectively,P<0.01)Conclusion Prokaryotic HAV capsid proteins VP1 and VP3 showed high purity and good immunogenicity,and the addition of CpG in the preparation was beneficial to enhance the immunogen

关 键 词：甲型肝炎病毒 基因重组 原核表达 衣壳蛋白 免疫原性 

分 类 号：R373.2[医药卫生—病原生物学]