表达SARS-CoV-2的RBD基因及多表位基因重组DNA候选疫苗的构建及小鼠免疫效果评价  

Construction of RBD vaccine and multi-epitope gene recombinant DNA vaccine candidate expressing SARS-CoV-2 and mouse immunization evaluation

作　　者：孙世宇 李卓昕[2] 哈卓 陈璐儿 刘宇梦 李晓宁 鲁会军[2] 金宁一 SUN Shi-yu;LI Zhuo-xin;HA Zhuo;CHEN Lu-er;LIU Yu-meng;LI Xiao-ning;LU Hui-jun;JIN Ning-yi(College of Animal Science and Veterinary Medicine,Guangxi University,Nanning 530004,China;Changchun Veterinary Research Institute,Chinese Academy of Agricultural Sciences)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]中国农业科学院长春兽医研究所

出　　处：《中国病原生物学杂志》2022年第1期1-4,8,共5页Journal of Pathogen Biology

基　　金：吉林省重点研发计划项目(No.20200901001SF);长春市发展计划项目(No.2020RW001)。

摘　　要：目的构建针对新型冠状病毒的重组核酸苗,评价其与佐剂联合免疫BALB/c小鼠的效果。方法选择新型冠状病毒S蛋白的受体结合域蛋白基因(RBD)和新冠病毒S、M、N、E 4个结构蛋白的抗原表位基因(EPI)为主要抗原基因,以pVAX1为载体,分别构建pVAX-RBD-EPI质粒和pVAX-RBD质粒。用2种重组质粒和pVAX空载体质粒分别与PIKCA佐剂联合免疫或无佐剂免疫BALB/c小鼠,共免疫3次,间隔21天免疫,每7 d对小鼠特异性抗体进行检测;对三免后21 d小鼠脾淋巴细胞进行T淋巴细胞亚类检测。结果成功构建了重组质粒pVAX-RBD-EPI和pVAX-RBD。用重组质粒转染BHK细胞,Western blot显示重组核酸质粒稳定表达目的蛋白。BALB/c小鼠免疫结果表明,三免后14d时pVAX-RBD-EPI+PIKCA组IgG抗体水平为无佐剂组的3.65倍(P<0.01)。三免后21 d,pVAX-RBD-EPI+PIKCA组CD3^(+)/CD4;T达到55.71%,是阴性对照组的1.52倍(P<0.01);三免后21 d,CD3^(+)/CD8^(+)T数量pVAX-RBD-EPI+PIKCA组为7.07%,是阴性对照组的1.83倍,是pVAX-RBD-EPI组(5.86%)的1.2倍(P<0.05)。结论成功构建重组DNA候选疫苗pVAX-RBD-EPI和pVAX-RBD-EPI。两种疫苗均具有良好的免疫原性,其中PIKCA免疫佐剂的加入可增强小鼠细胞免疫和体液免疫水平。Objective To construct the recombinant nucleic acid vaccine for novel coronavirus to evaluate the effect of immune BALB/c mice with adjuvant.Methods In this experiment,by selecting the receptor binding domain protein gene(RBD)of the COVID-19 S protein and the antigen epitope gene(EPI)of the four S,M,N,E structural proteins of COVID-19 as the primary study sites of pVAX1 vector.And the pVAX-RBD-EPI plasmid and the pVAX-RBD plasmid were constructed with the connection of pVAX1 and target gene,respectively.These two recombinant plasmids with adjuvant or not were immuned to BALB/c mice respectively.Specific antibodies were tested in mice every 7 days,and T lymphocyte subclasses were tested for mouse spleen lymphocytes for exemptiot on 21 d after third inoculation.Results Recombinant DNA plasmid pVAX-RBD-EPI and pVAX-RBD were successfully constructed.By transfecting with BHK cells with recombinant plasmids,Western blot showed recombinant DNA plasmid stably expresses the target protein.After 14 days of third immunization,IgG antibody levels in the pVAX-RBD-EPI+PIKCA group were 3.65 times the levels of the adjuvant-free group(P<0.01).After 63 days of immunization,the CD3^(+)/CD4;T of the pVAX-RBD-EPI+PIKCA group reached 55.71%,which was 1.52 times of the negative control group.After 21 days of third immunization,CD3^(+)/CD8^(+)T amount of the pVAX-RBD-EPI+PIKCA group reached 7.07%,1.83 times of the negative control group and 1.2 times of the pVAX-RBD-EPI group(5.86%)(P<0.05).Conclusion Recombinant DNA candidate vaccine pVAX-RBD-EPI and pVAX-RBD were successfully constructed.All DNA vaccines showed good immunogenicity,among which PIKCA immunologic adjuvant could induce the cellular immunity and humoral immunity in mice.

关 键 词：新型冠状病毒 受体结合域蛋白 抗原表位蛋白 免疫原性 

分 类 号：R373.1[医药卫生—病原生物学]