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 immunogenicity of antigens.This study laid a foundation of the development of HAV recombinant subunit vaccine.
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)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
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)。
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
Hepatitis A virus(HAV)
Gene recombination
Prokaryotic expression
Capsid protein
Immunogenicity
