| 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2 d。【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。 |
| 关键词: 隐孢子虫 Cp16基因 真核表达质粒 免疫保护性 |
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| 基金项目:国家科技支撑计划项目（20007BAD40B05）；国家“863”计划重点支持项目（2006AA10A207） |
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| Construction of eukaryotic expression vector of Cryptosporidium parvum Cp16 gene and its immunoprotective effect |
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| Abstract: |
| 【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 1×106 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. |
| Key words: C.parvum Cp16 gene eukaryotic expression plasmid immunoprotectiveness |
