刚地弓形虫SRS29C核酸疫苗的构建及SRS29C与SAG1联合免疫保护性的比较研究  

Construction of Toxoplasma gondi SRS29C nucleic acid vaccine and comparative study of the immunoprotection of SRS29C and SAG1 combination

作　　者：叶建军 田静 高传亮 闫安 叶利颖 王宇冰 宋淇淇 YE Jianjun;TIAN Jing;GAO Chuaniang;YAN An;YE Liying;WANG Yubing;SONG Qiqi(Tianjin City Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300392,China)

机构地区：[1]天津农学院动物科学与动物医学学院天津市农业动物繁育与健康养殖重点实验室,天津300392

出　　处：《黑龙江畜牧兽医》2023年第21期18-25,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：天津市自然科学基金项目(19JCQNJC13700);天津市教委科研计划项目(2018KJ185);国家级大学生创新训练计划项目(202110061035)。

摘　　要：为了探究刚地弓形虫表面蛋白(surface antigen,SAG)SRS29C基因和SRS29C、SAG1联合基因的核酸疫苗对小鼠急性感染弓形虫的免疫保护效果,试验利用PCR方法扩增了SRS29C基因片段并将其克隆至真核表达载体pEGFP-N1中,构建重组质粒pEGFP-SRS29C,并对该质粒进行酶切鉴定,同时用该质粒对HEK293T细胞进行转染,再用Western-blot方法检测目标蛋白在细胞中的表达情况;用重组质粒pEGFP-SRS29C和pEGFP-SAG1单独免疫或联合免疫小鼠,用ELISA法测定血清IgG水平,CCK-8法检测脾淋巴细胞增殖能力,流式细胞仪测定CD4^(+)和CD8^(+)T淋巴细胞百分比,ELISA试剂盒检测脾脏细胞因子的表达情况;进行小鼠体内攻虫试验,记录小鼠存活时间。结果表明:成功构建了重组质粒pEGFP-SRS29C,表达的蛋白质大小为66 ku;pEGFP-SRS29C组和联合免疫组与PBS组和空载体组相比血清IgG含量、脾淋巴细胞增殖率、CD4^(+)/CD8^(+)值均显著提高(P<0.05);pEGFP-SRS29C组和联合免疫组γ干扰素(IFN-γ)和肿瘤坏死因子-α(TNF-α)含量明显提高;联合免疫组诱免疫球蛋白G(IgG)、IFN-γ和TNF-α含量均高于pEGFP-SRS29C组和pEGFP-SAG1组。PBS组和空载体组小鼠存活8~9 d;pEGFP-SRS29C组小鼠存活(18.10±1.37)d,pEGFP-SAG1组小鼠存活(21.20±0.97)d,联合免疫组小鼠存活(24.20±1.72)d,极显著长于PBS组和空载体组(P<0.01)。说明单独使用构建的弓形虫核酸疫苗pEGFP-SRS29C或与pEGFP-SAG1联合使用能够诱导小鼠产生体液免疫和细胞免疫应答,增强小鼠抵抗弓形虫感染的能力,并且SRS29C作为保护性抗原与SAG1联合免疫的效果更好。In order to explore the immunoprotective effect of DNA vaccines with surface antigen(SAG)SRS29C gene,SRS29C and SAG1 combination genes on acute infection with Toxoplasma gondi in mice,in this experiment,the SRS29C gene fragments were amplified by PCR and cloned into the eukaryotic expression vector pECFP-N1,and the recombinant plasmid pECFP-SRS29C was constructed.The plasmid was identified by digestion and transfected with HEK293T cells;the Western-blot method was used to detect expression of target protein in cells.Recombinant plasmids pECFP-SRS29C and pEGFP-SAC1 were immunized in mice alone or in combination;the level of IgG in serum was measured by ELISA method,and the proliferation ability of splenic lymphocytes was detected by CCK-8 method.Flow cytometry measured the percentage of CD4^(+)and CD8^(+)Tlymphocytes,and the expression ofspleen cytokines was detected using ELISA kits.In vivo Toxoplasma gondi challenge test was conducted in mice,and mouse survival time was recorded.The results showed that the recombinant plasmid pECFP-SRS29C was successfully constructed,and the protein size expressed was 66 ku.Compared with PBS group and empty vehicle group,the serum IgG content,splenic lymphocyte proliferation rate and CD4^(+)/CD8^(+)values in pECFP-SRS29C group and combined immunization group were significantly increased(P<0.05).The contents of IFN-γ and TNF-αin pEGFP-SRS29C group and combined immunization group were significantly increased.The contents of IgG,IFN-γ and TNF-αwere higher in the combined immunization group than in the pEGFP-SRS29C group and pECFP-SAGI group.Mice in PBS group and empty vehicle group survived for 8-9 days;mice in the pEGFP-SRS29C group survived for(18.10±1.37)days;mice in the pECFP-SAG1 group survived for(21.20±0.97)days;mice in the combined immunization group survived for(24.20±1.72)days,which was significantly longer than the PBS group and the empty vehicle group(P<0.01).The results suggested that the constructed Toxoplasma gondi DNA vaccine pEGFP-SRS29C alone or in combinat

关 键 词：弓形虫 核酸疫苗 SRS29C SAG1 重组质粒 联合免疫 

分 类 号：S852.729[农业科学—基础兽医学]