| 隐孢子虫鼠基因型CP15基因真核表达质粒的构建及在Hela细胞中的表达 |
| |
| 引用本文： | 苏庆美,何生虎,米荣升,张国恩,黄燕,周鹏,秦培兰,呼高伟,陈兆国. 隐孢子虫鼠基因型CP15基因真核表达质粒的构建及在Hela细胞中的表达[J]. 中国兽医科学, 2011, 0(5) |
| |
| 作者姓名： | 苏庆美 何生虎 米荣升 张国恩 黄燕 周鹏 秦培兰 呼高伟 陈兆国 |
| |
| 作者单位： | 宁夏大学农学院;中国农业科学院上海兽医研究所农业部动物寄生虫学重点开放实验室中国农业科学院动物源性食品安全研究中心; |
| |
| 基金项目： | 国家“十一五”高技术研究发展计划(863)项目(2006AA10A207);国家“十一五”科技支撑计划项目(2007BAD40B05); 上海市科技兴农重点攻关项目[沪农科攻字(2005)第3-4号]; 中央级公益性科研院所基本科研业务费专项资金项目(2010JB12) |
| |
| 摘 要： | 为了构建隐孢子虫鼠基因型CP15基因的重组真核表达质粒,检测其重组质粒在Hela细胞中的表达。采用PCR方法,从pMD18-T-CP15菌液中扩增了CP15及含寡聚脱氧核苷酸(CpG-ODN)的CP15基因,酶切测序鉴定;将该基因亚克隆至pVAX1载体中,用脂质体介导的方法转染Hela细胞,RT-PCR法、间接免疫荧光法和Western-blot法检测表达蛋白的生物学活性。结果显示,扩增了约360 bp和380 bp的隐孢子虫鼠基因型CP15和含CpG-ODN的CP15基因,酶切测序鉴定成功构建了pVAX1-CP15和含有CpG-ODN的重组真核表达质粒pVAX1-C-CP15。转染Hela细胞后,用RT-PCR法检测到外源基因有效的转录,用间接免疫荧光、SDS-PAGE和Western-blot法检测到外源基因的有效表达。结果表明,构建的重组真核表达质粒能够在Hela细胞中成功转录和表达,并且表达产物具有良好的免疫活性。这为下一步深入研制具有高保护性的隐孢子虫核酸疫苗奠定了基础。
|
| 关 键 词： | 隐孢子虫鼠基因型 CP15 真核表达质粒 Hela细胞 表达 |
Construction of eukaryotic expression plasmids with Cryptosporidium mouse genotype CP15 gene and their expression in Hela cells |
| |
| SU Qing-mei,,HE Sheng-hu,MI Rong-sheng,ZHANG Guo-en,HUANG Yan,ZHOU Peng,QIN Pei-lan,HU Gao-wei,CHEN Zhao-guo. Construction of eukaryotic expression plasmids with Cryptosporidium mouse genotype CP15 gene and their expression in Hela cells[J]. Chinese Veterinary Science, 2011, 0(5) |
| |
| Authors: | SU Qing-mei HE Sheng-hu MI Rong-sheng ZHANG Guo-en HUANG Yan ZHOU Peng QIN Pei-lan HU Gao-wei CHEN Zhao-guo |
| |
| Affiliation: | SU Qing-mei1,2,HE Sheng-hu1,MI Rong-sheng2,ZHANG Guo-en2,HUANG Yan2,ZHOU Peng2,QIN Pei-lan2,HU Gao-wei2,CHEN Zhao-guo2(1.College of Agriculture,Ningxia University,Yinchuan 750021,China,2.Animal-borne Food Safety Research Center of Chinese Academy of Agricultural Sciences/Key Laboratory of Animal Parasitology of the Ministry of Agriculture/Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China) |
| |
| Abstract: | In order to construct recombinant expression plasmids with Cryptosporidium mouse genotype CP15 gene and express them in Hela cell,CP15 gene and CP15 gene containing CpG-ODN(C-CP15) were amplified from pMD18-T-CP15 by PCR.The CP15 gene and C-CP15 gene were identified by enzyme digestion and sequencing.Then the CP15 gene and C-CP15 gene were cloned into the pVAX 1 vector.Then Hela cells were transfected with recombinant expression plasmids by Lipofectarnine and assayed by RT-PCR,indirect immunofluorescence,SD... |
| |
| Keywords: | Cryptosporidium mouse genotype CP15 eukaryotic expression plasmid Hela cell expression |
| 本文献已被 CNKI 等数据库收录！ |
|
