| 非洲马瘟病毒VP7基因的克隆及其在昆虫细胞中的表达 |
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| 引用本文： | 李富祥,杨仕标,艾军,周晓黎,赵文华,王金萍.非洲马瘟病毒VP7基因的克隆及其在昆虫细胞中的表达[J].中国畜牧兽医,2012,39(1):28-31. |
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| 作者姓名： | 李富祥 杨仕标 艾军 周晓黎 赵文华 王金萍 |
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| 作者单位： | 1. 云南省畜牧兽医科学院,云南昆明 650224;2. 云南出入境检验检疫局,云南昆明 650228 |
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| 基金项目： | 公益性行业(农业)科研专项 |
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| 摘 要： | 为了获得具有天然活性的非洲马瘟病毒重组VP7蛋白,制备针对非洲马瘟VP7蛋白的单克隆抗体及建立快速抗体检测方法,本试验通过PCR扩增、胶回收纯化后经Xba Ⅰ和Hind Ⅲ特异性酶切,将VP7基因克隆于昆虫杆状病毒表达载体pFastBacHTB,经PCR、酶切、测序鉴定后,成功构建了携带VP7基因的重组质粒pFastBacHTB-AHSV-VP7。该重组质粒转化含有杆状病毒穿梭载体的DH10Bac感受态细胞,经抗生素、PCR筛选,获得转座的杆粒Bacmid-AHSV-VP7,并在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒,再感染Sf9昆虫细胞,收获表达产物。表达产物经Western blotting分析表明,VP7重组蛋白得到表达,其分子质量大小约为45 ku,且具有良好的生物活性。
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| 关 键 词： | 非洲马瘟病毒 VP7基因 杆状病毒表达 昆虫细胞 |
| 收稿时间： | 2011-06-21 |
Cloning and Expression of VP7 Gene of African Horse Sickness Virus in Baculovirus Infected Insect Cells |
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| LI Fu-xiang
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YANG Shi-biao
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AI Jun
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ZHOU Xiao-li
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ZHAO Wen-hua
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WANG Jin-ping.Cloning and Expression of VP7 Gene of African Horse Sickness Virus in Baculovirus Infected Insect Cells[J].China Animal Husbandry & Veterinary Medicine,2012,39(1):28-31. |
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| Authors: | LI Fu-xiang YANG Shi-biao AI Jun ZHOU Xiao-li ZHAO Wen-hua WANG Jin-ping |
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| Institution: | 1. Yunnan Animal Science and Veterinary Institute, Kunming 650224,China;2. Yunnan Entry-Exit Inspection and Quarantine Bureau,Kunming 650228,China |
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| Abstract: | The VP7 gene fragment was cloned to the vector pFastBacHTB.The recombinant plasmid pFastBacHTB-AHSV-VP7 was constructed and was identified by PCR and enzyme digestion and sequenced.Then the plasmid pFastHTB-AHSV-VP7 was transformed into DH10Bac complement cells.It was identified by antibiotics and PCR,it showed the recombinant plasmid pFastBacHTB-AHSV-VP7 was constructed.On this basis,transposition bacmid DNA was extracted to transfect Sf9 insect cells.After transfected 96 hours,baculovirus was harvested.Then the Western blotting showed that the recombinant VP7 was expressed in insecet cells.The recombinant VP7 protein had a good biological activity and was approximate 45 ku.It is a base to construct a ELISA kit to test AHSV antibody and to prepare monoclonal antibody. |
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| Keywords: | African horse sickness virus(AHSV) VP7 gene baculovirus expression insect cells |
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