弓形虫SAG1、ROP18真核表达载体的构建及在诱导小鼠保护性免疫中的作用研究  

Construction of eukaryotic expression vectors of Toxoplasma gondii SAG1 and ROP18 and their role in inducing protective immunity in mice

作　　者：吴腊梅 彭天丽 何雁鸿 董成林 杨慧健 WU Lamei;PENG Tianli;HE Yanhong;DONG Chenglin;YANG Huijian(Department of Clinical Laboratory,Anting Hospital,Jiading District,Shanghai 201800,China;Department of Clinical Laboratory,Shanghai Zhongye Hospital,Shanghai 200941,China)

机构地区：[1]上海市嘉定区安亭医院检验科,上海201800 [2]上海中冶医院检验科,上海200941

出　　处：《国际检验医学杂志》2023年第19期2365-2369,共5页International Journal of Laboratory Medicine

基　　金：上海市卫生和健康委员会青年基金项目(20184Y0087);上海市嘉定区自然科学基金项目(JDKW-2022-0052)。

摘　　要：目的观察弓形虫速殖子表面抗原1(SAG1)、棒状体蛋白18(ROP18)真核表达载体单独及联合使用诱导BALB/c小鼠的保护性免疫作用。方法制备重组真核表达质粒p3×FLAG-Myc-CMV TM-24-SAG1,p3×FLAG-Myc-CMV TM-24-ROP18及空质粒p3×FLAG-Myc-CMV TM-24(空载体),以磷酸盐缓冲液(PBS)稀释至1μg/μL。将102只BALB/c小鼠随机分成6组,每组17只,前4组分别注射PBS(PBS对照组)、空载体(空载体对照组)、SAG1载体(SAG1组)、ROP18载体(ROP18组)各100μL,其余两组均注射SAG1与ROP18对半混合载体,分别注射量为200μL(全量混合组)、100μL(半量混合组),每组间隔2周免疫1次,共免疫3次。末次免疫4周后,各组随机取7只小鼠,无菌条件下取脾,流式细胞术检测小鼠脾脏CD4^(+)、CD8^(+)T细胞亚群分布,荧光定量PCR分析各组小鼠脾细胞中细胞因子白细胞介素(IL)-4、IL-12、干扰素γ(IFN-γ)的表达;每组其余10只小鼠腹腔接种1×103个弓形虫速殖子,观察各组小鼠的生存时间。结果各组小鼠脾脏CD4^(+)T细胞比例差异无统计学意义(P>0.05);与PBS对照组比较,SAG1组、ROP18组、全量混合组、半量混合组、空载体对照组CD8^(+)T细胞比例明显升高(P<0.01),半量混合组较其他组更高(P<0.05)。半量混合组、全量混合组CD4^(+)/CD8^(+)较两对照组(PBS对照组和空载体对照组)降低,差异有统计学意义(P<0.05)。全量混合组、半量混合组小鼠脾细胞中IL-12、IFN-γ相对表达水平与两对照组比较,差异有统计学意义(P<0.05),且半量混合组IFN-γ相对表达水平与ROP18组比较,差异有统计学意义(P<0.05),但各组间IL-4相对表达水平差异无统计学意义(P>0.05)。弓形虫速殖子攻击感染小鼠试验中两单基因疫苗组(SAG1组和ROP18组)、全量混合组、半量混合组均较两对照组生存时间延长(P<0.05),且半量混合组较全量混合组生存时间也显著延长(P<0.01),但全量混合组与两单基因疫苗组差异无统计学意义(P>0.05)Objective To observe the protective immunity function of BALB/c mice induced by the eukaryotic expression vectors of SAG1 and ROP18 of Toxoplasma gondii alone or in combination.Methods Recombinant eukaryotic expression plasmid p3×FLAG-Myc-CMV TM-24-SAG1,p3×FLAG-Myc-CMV TM-24-ROP18 and empty plasmid p3×FLAG-Myc-CMV TM-24(empty vector)were prepared and diluted to 1μg/μL with phosphate buffer solution(PBS).A total of 102 BALB/c mice were randomly divided into six groups,with 17 mice per group.The first four groups were injected with 100μL of PBS(PBS control group),empty vector(empty vector control group),SAG1 vector(SAG1 group)and ROP18 vector(ROP18 group)respectively,and another two groups were injected with the half-mixed vector of SAG1 and ROP18,and the injection dose were 200μL(full mixed group)and 100μL(half mixed group)respectively.Each group was immunized once two weeks,three times in total.Four weeks after the final immunization,7 mice were randomly selected from each group,and their spleen was taken aseptically.The distribution of CD4^(+)and CD8^(+)T cell subsets in the spleen of mice was detected by flow cytometry,and the mRNA expression of cytokines interleukin(IL)-4,IL-12 and interferonγ(IFN-γ)in each group was analyzed by fluorescence quantitative PCR.The remaining ten mice in each group were intraperitoneally inoculated with 1×103 Toxoplasma gondii tachyzoites,and the survival time of each group was observed.Results There was no significant difference in the proportion of CD4^(+)T cells in spleen among all groups(P>0.05).Compared with PBS group,the proportion of CD8^(+)T cells in SAG1 group,ROP18 group,full mixed group,half mixed group and empty vector control group significantly increased(P<0.01),and the proportion of CD8^(+)T cells in half mixed group was higher than that in other groups(P<0.05).In the half mixed group and the full mixed group,the CD4^(+)/CD8^(+)was lower than those of the two control groups,which were PBS control group and empty vector control group,and the difference was

关 键 词：刚地弓形虫 速殖子表面抗原1 棒状体蛋白18 DNA疫苗 

分 类 号：R382.5[医药卫生—医学寄生虫学] R446.6[医药卫生—基础医学]