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口蹄疫病毒P1 2A和3C基因重组逆转录
病毒表达载体的构建
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刘艳红1，李炯1 ，刘俊林2，刘湘涛1，尚佑军1，殷宏1
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(1.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
开放实验室，甘肃 兰州730046； 2.甘肃农业大学 动物医学院，甘肃 兰州730070)
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摘要：
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以A型口蹄疫病毒(FMDV)AV88(L)和XJ99株的P1 2X基因和AV88(L) 3C基因的阳性克隆质粒为模板,用PCR扩增各基因片段，酶切后分步定向亚克隆至逆转录病毒表达载体pBABE-puro上经PCR、酶切鉴定及测序。结果表明，获得了2个含不同RGD基序的A型FMDV衣壳蛋白和蛋白酶基因的重组逆转录病毒表达载体pBABE-AV88(L) P1 2X3C和pBABE-XJ99 P1 2X3C，为研究安全、高效、低成本的FMDV空衣壳亚单位疫苗奠定了基础。 |
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关键词：
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A型口蹄疫病毒；衣壳蛋白基因；蛋白酶基因；重组逆转录病毒表达载体 |
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中图分类号：S 852.659.6文献标识码：
A 文章编号：1673-4696(2006)08-0606-05 |
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Construction of the recombinant retroviral vector with P1 2A
and 3C genes of FMDV |
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LIU Yan-hong1,LI Jiong1,LIU
Jun-lin2,LIU Xiang-tao1,SHANG You-jun1,YIN
Hong1 |
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(1.Key Laboratory of Aminal Virology,Ministry of Agriculture/State Key Laboratory of Veterinary
Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,
Lanzhou 730046,China；2.College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China) |
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Abstract: By using two-step cloning strategy,the fragment P1 2X3C of foot and mouth disease virus serotype A was obtained and two recombinant retrovirus expression vectors pBABE-AV88(L) P1 2X3C and pBABE-XJ99 P1 2X3C were constructed.P1 2A and 3C genes with flanking restriction sites for endonuclease were amplified respectively by PCR,and then the P1 2X gene was digested by BamHⅠ and EcoRⅠ.The gene P1 2X was cloned into the pBABE expression vector which was digested by the same enzyme.The recombinant plasmid was identified by PCR,digestion with BamHⅠ + EcoRⅠand sequencing.After the P1 2X plasmid was constructed,the gene 3C flanked EcoRⅠ was cloned into the P1 2X positive expression plasmid.The result indicated that the fragment P1 2X3C was cloned into the retrovirus expression plasmid correctly. |
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Key words: foot-and-mouth disease virus type A; recombinant retrovirus expression vector; capsid protein gene; proteinase gene |
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