微小隐孢子虫Cp16基因真核表达载体的构建及其免疫保护性研究  

Construction of eukaryotic expression vector of Cryptosporidium parvum Cp16 gene and its immunoprotective effect

作　　者：肖丹[1] 殷驰[1] 李建华[1] 宫鹏涛[1] 张西臣[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《西北农林科技大学学报（自然科学版）》2010年第12期25-30,37,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家科技支撑计划项目(20007BAD40B05);国家"863"计划重点支持项目(2006AA10A207)

摘　　要：【目的】构建微小隐孢子虫黏附蛋白相关基因Cp16的真核表达载体pVAX1-Cp16,并检测其对小鼠的免疫保护效果。【方法】用PCR方法扩增微小隐孢子虫Cp16基因,构建其真核表达载体pVAX1-Cp16,并用脂质体介导法将其转染Hela细胞,用间接免疫荧光技术、SDS-PAGE及Western-blot检测Cp16蛋白在转染细胞中的表达情况;分别用真核表达载体pVAX1-Cp16、pVAX1空载体及PBS肌肉注射免疫BAILB/c小鼠,共免疫3次,检测血清IgG抗体水平及CD4+和CD8+T细胞亚群的变化,并用微小隐孢子虫卵囊感染免疫小鼠(剂量为1×106个/只),研究真核表达载体pVAX1-Cp16对隐孢子虫感染小鼠的免疫保护作用。【结果】成功构建了pVAX1-Cp16真核表达载体;Western-blot和间接免疫荧光检测结果显示,Cp16基因可以在Hela细胞中表达,且表达产物具有反应原性。pVAX1-Cp16免疫小鼠(试验组)血清中抗体水平及CD4+与CD8+T细胞数目比值升高,与各对照组(pVAX1和PBS对照组)相比差异显著(P<0.05)。各组小鼠经口感染微小隐孢子虫卵囊后,试验组与各对照组相比,卵囊排出量均减少了63.97%,持续排出卵囊时间缩短了2d。【结论】构建了微小隐孢子虫黏附蛋白相关基因Cp16的真核表达载体pVAX1-Cp16,其能刺激机体产生一定的免疫保护作用。[Objective] In this study,the eukaryotic vector pVAX1-Cp16 was construced by PCR.and its in vivo efficacies were tested in BALB/c mice.[Method] The DNA sequence of Cryptosporidium parvum adhension gene（Cp16）was amplified and then cloned into pVAX1 vector to form the recombinant plasmids pVAX1-Cp16.Hela cells were transfected by plasmid pVAX1-Cp16 with lipofection strategy.Recombinant protein expression from the plasmid in HeLa cells was confirmed by indirect immunofluorescence staining and Western-blot analysis.The plasmid pVAX1-Cp16,pVAX1 and PBS was respectively used to immunize BALB/c mouse by injection in thigh muscle.Two weeks after the third immunization,the humoral and cellular responses BAILB/c in each group were tested and approximately challenged with 1×10^6 oocysts to observe the immunoprotective efficacy.[Result] The results showed that the Cp16 protein has been successfully expressed in Hela cells which was detected by Immunofluorescence staining or Western-blot with coat anti-C.parvum serum.The results indicated that the group of pVAX1-Cp16 elicited significant antibody responses and specific cellular responses when compared to control mice that received vector only or PBS（P〈0.05）.The experiment that the BALB/c mice were inculated with C.parvum oocysts showed pVAX1-Cp16 had immuno-protective effect on C.parvum infection in mice.The amount of evacuated oocysts in experiment group was decreased by 63.97%,Oocyst discharge shortened by two days.[Conclusion] The eukaryotic vector pVAX1-Cp16 was constructed and it could provide partial immune protection against Cryptosporidium parvum challenge.

关 键 词：隐孢子虫 Cp16基因 真核表达质粒 免疫保护性 

分 类 号：S852.723[农业科学—基础兽医学]