弓形虫ROPs DNA疫苗对小鼠急性感染弓形虫的免疫保护作用  

Immunoprotective effects of Toxoplasma gondi ROPs DNA vaccine on acute infection with Toxoplasma gondi in mice

作　　者：杜荣起 李丹若 何金灵 龙芯 赵瑞利 赵伟 金天明 张东超 DU Rongqi;LI Danruo;HE Jinling;LONG Xin;ZHAO Ruili;ZHAO Wei;JIN Tianming;ZHANG Dongchao(College of Animal Science and Veterinary Medicine/Tianjin City Key Laboratory of Agricultural Animal Breeding and Healthy Husbandry,Tianjin Agricultural University,Tianjin 300392,China;Tianjin Academy of Agricultural Sciences,Tianjin 300192,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 [2]天津市农业科学院,天津300192

出　　处：《黑龙江畜牧兽医》2023年第21期11-17,25,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金青年基金项目(32202760);天津市农业动物繁育与健康养殖重点实验室开放基金项目(2020zdkf02);天津市研究生科研创新项目(2021YJSS137);天津市教委科技计划项目(2019KJ032);天津市生猪产业技术体系创新团队项目(ITTPRS2021005)。

摘　　要：为了评价弓形虫棒状体蛋白(ROPs)DNA疫苗对小鼠急性感染弓形虫的免疫保护作用,试验将获取的ROP5、ROP17和ROP18基因分别与pVAX1载体连接,获得重组质粒pVAX1-ROP5、pVAX1-ROP17和pVAX1-ROP18。将重组质粒转染至HEK293A细胞中,利用Western-blot方法检测目的蛋白在真核细胞内的表达情况。将获得的重组质粒等摩尔比混合后通过腿部肌肉注射免疫小鼠作为混合疫苗组,同时设置空载体组及生理盐水组,免疫后检测小鼠血清IgG抗体水平、脾淋巴细胞增殖情况、细胞因子水平及T淋巴细胞分型,加强免疫后第14天对小鼠腹腔接种弓形虫RH株并观察其生存情况。结果表明:重组质粒经PCR扩增、测序及双酶切验证,分别在1686,1863,1701 bp处出现特异性条带,与目的基因大小一致且无碱基增加、缺失和突变情况;重组质粒经Western-blot检测在62,68,63 ku处成功表达;与空载体组、生理盐水组相比,混合疫苗组血清IgG抗体水平、脾淋巴细胞增殖指数、IFN-γ和IL-6细胞因子、CD4^(+)T淋巴细胞占比和CD4^(+)/CD8^(+)均极显著升高(P<0.01),免疫小鼠攻虫后的生存时间[(18.4±1.4)d]极显著延长(P<0.01)。说明构建的弓形虫ROPs DNA疫苗pVAX1-ROP5/ROP17/ROP18能够刺激小鼠产生高水平的体液免疫和细胞免疫应答,并能增强小鼠抵抗弓形虫感染的能力。In order to evaluate the immunoprotective effects of Toxoplasma gondi rhoptry proteins(ROPs)DNA vaccine on acute infection with Toxoplasma gondi in mice,in this experiment,the acquired ROP5,ROP17 and ROP18 genes were ligated to the pVAXI vector respectively to obtain recombinant plasmids pVAX1-ROP5,pVAX1-ROP17 and pVAX1-ROP18.The recombinant plasmids were transfected into HEK293A cells and the Western-blot method was used to detect the expression of the target protein in eukaryotic cells.The obtained recombinant plasmids were mixed with equal molar ratio and were immunized to mice by leg muscle injection as the mixed vaccine group,and the Empty carrier group and normal saline group were set up at the same time.After immunization,mice were detected for serum IgG antibody level,splenic lymphocyte proliferation,cytokine level and T lymphocyte typing.Toxoplasma gondi RH strain was inoculated on day 14 after immunization and the mice were observed for survival.The results showed that the recombinant plasmids were amplified by PCR,sequenced and doubledigested,and specific bands appeared at686,8631p,respectively,which were consistent with the sizesofthetarget genes and had no base addition,deletion and mutation.The recombinant plasmids were successfully expressed at 62,68,63 ku by Westem blot detection.Compared with the empty carrier group and normal saline group,serum IgG antibody,spleen lymphocyte proliferation index,IFN-and IL-6 cytokines,CD4^(+)T lymphocyte proportion and CD4^(+)/CD8^(+)ratio were very significantly increased in the mixed vaccine group(P<0.01).The survival time([18.4±1.4]d)of the vaccinated mice after Toxoplasma gondi challenge was significantly prolonged(P<0.01).The results suggested that the constructed Toxoplasma gondi ROPs DNA vaccine pVAX1-ROP5/ROP17/ROP18 could stimulate mice to produce high levels of humoral and cellular immune responses and could enhance resistance ability of mice to Toxoplasma gondinfection.

关 键 词：弓形虫 DNA疫苗 ROP5 ROP17 ROP18 

分 类 号：S855.99[农业科学—临床兽医学]