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口蹄疫病毒2C′3AB基因重组杆状病毒的构建及其表达
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张小丽1，2,卢曾军2,田美娜2 ,孙普2,曹轶梅2，付元芳1，2，马小军1，
刘在新2*,谢庆阁2
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（1．甘肃农业大学 动物医学院，甘肃 兰州730070；2．中国农业科学院 兰州兽医研究所 家畜疫病病原
生物学国家重点实验室 农业部畜禽病毒学重点实验室 国家口蹄疫参考实验室，甘肃 兰州730046）
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摘要：
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将含有口蹄疫病毒(FMDV)非结构蛋白2C部分基因编码区及3AB完整基因编码区的基因片段2C′3AB重组于杆状病毒穿梭质粒pMelBac-B，经PCR、酶切及序列分析鉴定正确后，命名为pMel-2C′3AB。将重组穿梭质粒pMel-2C′3AB与杆状病毒骨架共转染Sf9昆虫细胞，通过噬斑筛选及PCR鉴定，构建了含有目的基因的重组杆状病毒。用该病毒感染High Five细胞，细胞裂解液的SDS-PAGE电泳结果显示，出现一约为43ku的蛋白带，与预期大小一致；Western-blotting结果表明，该表达产物能被FMDV阳性血清及2C′3AB单克隆抗体识别；间接ELISA结果显示，表达的目的蛋白能与牛、羊、猪的O型FMDV阳性血清发生特异性反应。证实，FMDV 2C′3AB基因在昆虫细胞中得到了表达，且表达产物具有良好的反应活性。 |
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关键词：
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口蹄疫病毒；2C′3AB基因；重组杆状病毒；表达 |
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中图分类号：S 852.659.6：Q 786 文献标识码：
A 文章编号：1673-4696(2008)06-0483-06 |
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Construction and expression of recombinant baculovirus with
2C′3AB gene of foot-and-mouth disease virus |
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ZHANG Xiao-li1,2,LU Zeng-jun2,TIAN
Mei-na2,SUN Pu2,CAO Yi-mei2,FU Yuan-fang1,2,
MA Xiao-jun1,LIU Zai-xin2,XIE Qing-ge2
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(1.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China；2.National Foot-and-Mouth
Disease Reference Laboratory/Key Laboratory of Animal Virology of the Ministry of Agriculture/State Key
Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy
of Agricultural Sciences,Lanzhou 730046,China) |
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Abstract: 2C′3AB genes encoding a part of the nonstructural protein 2C and whole 3AB protein of foot-and-mouth disease virus(FMDV) were subcloned into a pMelBac-B shuttle plasmid.After identification by PCR,enzymatic digestion,and sequencing,a target recombinant plasmid was designated as pMel-2C′3AB.Then,the pMel-2C′3AB plasmid and linearized Bac-N-B1ueTM DNA were co-transfected into Sf9 insect cells mediated by Cellfectin reagent.Recombinant baculoviruses were screened by plaque assay and pure virus clone only containing the 2C′3AB genes only was identified by PCR.The High Five cells were infected with the recombinant baculovirus and selected for the expression of 2C′3AB protein.The lysates of the cells were analyzed by SDS-PAGE and Western-blotting.SDS-PAGE analysis showed that 2C′3AB protein of 43ku in molecular mass was expressed successfully,and it could be recognized specifically by FMDV positive serum and 2C′3AB McAb in the Western-blotting analysis.An indirect ELISA indicated that the expressed 2C′3AB was able to react specifically with FMDV positive serum from cattle,pig and sheep.The results confirmed that the 2C′3AB genes was expressed in Sf9 cells and that the expressed product had good reactogenicity. |
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Key words: foot-and-mouth disease virus；2C′3AB gene;recombinant baculovirus；expression |
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