刚地弓形虫棒状体蛋白18和膜表面抗原30复合核酸疫苗对小鼠的免疫保护作用  

Immunoprotection of nuclei acid vaccine dual⁃targeting rhoptry protein 18 and surface antigen 30 of Toxoplasma gondii in mice

作　　者：姜文静[1] 孟雅莉 赵利娜 王春苗[1] 张晓磊 JIANG Wenjing;MENG Yali;ZHAO Lina;WANG Chunmiao;ZHANG Xiaolei(College Lab Medicine,Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院医学检验学院,张家口075000

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第5期532-538,共7页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：河北省高等学校科学技术研究项目(2021013);河北省大学生创新创业训练计划(202155)。

摘　　要：目的研究刚地弓形虫棒状体蛋白18(ROP18)和膜表面抗原30(P30)复合核酸疫苗的免疫保护作用。方法PCR扩增弓形虫p30和rop18基因,构建pVAX1⁃p30、pVAX1⁃rop18⁃p30质粒并进行PCR鉴定、酶切鉴定、测序鉴定。用LipofectamineTM 3000转染试剂将pVAX1(2μg)和pVAX1⁃rop18⁃p30质粒转染至HeLa细胞内,24 h后间接免疫荧光法检测目的蛋白的表达情况。75只雌性BALB/c小鼠随机分为pVAX1⁃rop18⁃p30组、pVAX1⁃rop18组、pVAX1⁃p30组、pVAX1组和PBS组,每组15只,分别于股四头肌注射100μg(100μl)的pVAX1⁃rop18⁃p30、pVAX1⁃rop18(本实验室保存)、pVAX1⁃p30、pVAX1质粒或PBS(100μl),共免疫3次,间隔2周,末次免疫时质粒剂量为200μg。每次免疫前和末次免疫后2周采集小鼠血样,ELISA检测血清IgG抗体水平。末次免疫后2周每组取3只小鼠,无菌取脾细胞,培养后ELISA检测培养上清中γ干扰素(IFN⁃γ)和白细胞介素2(IL⁃2)、IL⁃4、IL⁃10、IL⁃12的水平。末次免疫后2周,每组取12只小鼠,每鼠腹腔注射1000个弓形虫RH株速殖子进行急性感染,记录小鼠存活时间,评价免疫保护性。组间比较采用单因素方差分析。结果弓形虫p30和rop18基因PCR扩增片段分别为789、1665 bp。pVAX1⁃rop18⁃p30质粒经PCR、酶切鉴定均获得预期大小的条带,测序鉴定结果正确。pVAX1⁃rop18⁃p30转染HeLa细胞后,胞质内可见黄绿色荧光,pVAX1转染的HeLa细胞内无黄绿色荧光。ELISA检测结果显示,末次免疫后2周,pVAX1⁃rop18⁃p30组吸光度(A450值)为0.788±0.025,高于pVAX1⁃rop18组(0.512±0.027)、pVAX1⁃p30组(0.498±0.027)、pVAX1组(0.122±0.014)和PBS组(0.109±0.011)(F=77.6,P<0.05),pVAX1⁃rop18组和pVAX1⁃p30组之间A450值差异无统计学意义(F=67.80,P>0.05);小鼠血清IgG抗体水平随着免疫次数的增加和时间延长而升高。末次免疫后2周,pVAX1⁃rop18⁃p30组小鼠脾细胞培养上清中IFN⁃γ、IL⁃2、IL⁃4、IL⁃10和IL⁃12的水平分�Objective To study the immunoprotective effect of a nucleic acid vaccine dual⁃targeting rhoptry protein 18(ROP18)and surface antigen 30(P30)of Toxoplasma gondii.Methods The p30 and rop18 genes of T.gondii were amplified by PCR to construct recombinant plasmids pVAX1⁃p30 and pVAX1⁃rop18⁃p30,which were veri⁃fied by PCR,enzyme digestion,and sequencing.HeLa cells were transfected with pVAX1 plasmids(2μg)and pVAX1⁃rop18⁃p30 plasmids using LipofectamineTM 3000 transfection reagents,and the expression of target protein was detected by indirect immunofluorescence assay after 24 hours.Seventy⁃five female BALB/c mice were randomly divided into five groups(pVAX1⁃rop18⁃p30 group,pVAX1⁃rop18 group,pVAX1⁃p30 group,pVAX1 group,and PBS group),with each group containing 15 mice.The plasmids[100μg(100μl)]and PBS(100μl)were injected into the quadriceps femoris muscle,with a total of three injections at two⁃week intervals,and the last injection had a plasmid dosage of 200μg.The levels of serum IgG were quantified by ELISA before each injection and two weeks after the final dose.The spleen cells were collected aseptically from three mice in each group two weeks after the last dose,and the levels of IFN⁃γ,IL⁃2,IL⁃4,IL⁃10,and IL⁃12 in the culture supernatant were measured by ELISA.Two weeks after the last immunization,12 mice were selected from each group,and 1000 tachyzoites of T.gondii RH strain were injected in⁃traperitoneally into each mouse to induce acute infections.The survival time of the mice was recorded,and their im⁃mune protection was evaluated.Single⁃factor analysis of variance was used for inter⁃group comparison.Results PCR amplification fragments of p30 and rop18 genes with lengths of 789 bp and 1665 bp.The pVAX1⁃rop18⁃p30 recombi⁃nant plasmids were successfully identified through PCR,enzyme digestion,and sequencing.HeLa cells transfected with pVAX1⁃rop18⁃p30 exhibited yellow⁃green fluorescence in the cytoplasm,while no such fluorescence was observed in cells

关 键 词：刚地弓形虫 棒状体蛋白18 膜表面抗原30 核酸疫苗 免疫保护 

分 类 号：R382.5[医药卫生—医学寄生虫学]