禽呼肠孤病毒σA蛋白在HEK293T细胞中的表达
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摘要
为了正确表达禽呼肠孤病毒(ARV)σA蛋白,试验拟构建σA基因的真核表达质粒,并将其在人胚肾细胞(HEK293T)中准确表达,参照GenBank中ARV S2基因序列(登录号为KF741763. 1)设计特异性引物,以pMD18-T-σA重组载体为模板,应用PCR的方法扩增σA基因的特异性序列,并将其克隆到p MD18-T载体中,构建重组pMD18-T-σA表达质粒,再用限制性内切酶KpnⅠ和NotⅠ同时双酶切pMD18-T-σA和真核表达质粒pEF1α-HA,将胶回收的σA基因和pEF1α-HA进行连接,构建真核表达质粒pEF1α-HA-σA。真核表达质粒经菌落PCR、双酶切和测序验证正确后,将其转染HEK293T细胞,于24 h后收取蛋白质,通过Western-blot验证目的蛋白。结果表明:本试验成功克隆了ARVσA基因,构建了其真核表达质粒pEF1α-HA-σA,并在HEK293T细胞中表达。说明该蛋白可以在HEK293T细胞中准确表达。
This study aimed to construct eukaryotic expression vector of Avian reovirus σAgene and express it accurately in HEK293 T cells. The specific primers of Avian reovirus σA gene were designed according to the gene sequence of Avian reovirus S2 gene from GenBank(accession No: KF741763. 1). pMD18-T-σA The Avian reovirus σA gene fragment was amplified from pMD18-T-σA,The specific sequence of σA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant vector.The cloning vector pMD18-T-σA and eukaryotic expression vector pEF1α-HA were double digested by the restriction enzyme KpnⅠ and NotⅠ. The σA and pEF1α-HA fragments were recycled,then the σA gene fragment were connected to pEF1α-HA to construct the recombinant vector pEF1α-HA-σA.And the recombinant vector pEF1α-HA-σA was identified by PCR,double enzyme digestion and sequencing. Then the recombinant vector p EF1α-HA-σA was tansfected into the HEK293 T cells,the protein was collected at 24 h after tansfection and verified by Western-blot. The result showed that the Avian reovirus σA gene was successfully cloned,and the eukaryotic expression vector pEF1α-HA-σA was successfully constructed,and the recombinant vector could expresse σA in HEK293 T cells. This indicates that the protein can be accurately expressed in HEK293 T cells.
This study aimed to construct eukaryotic expression vector of Avian reovirus σAgene and express it accurately in HEK293 T cells. The specific primers of Avian reovirus σA gene were designed according to the gene sequence of Avian reovirus S2 gene from GenBank(accession No: KF741763. 1). pMD18-T-σA The Avian reovirus σA gene fragment was amplified from pMD18-T-σA,The specific sequence of σA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant vector.The cloning vector pMD18-T-σA and eukaryotic expression vector pEF1α-HA were double digested by the restriction enzyme KpnⅠ and NotⅠ. The σA and pEF1α-HA fragments were recycled,then the σA gene fragment were connected to pEF1α-HA to construct the recombinant vector pEF1α-HA-σA.And the recombinant vector pEF1α-HA-σA was identified by PCR,double enzyme digestion and sequencing. Then the recombinant vector p EF1α-HA-σA was tansfected into the HEK293 T cells,the protein was collected at 24 h after tansfection and verified by Western-blot. The result showed that the Avian reovirus σA gene was successfully cloned,and the eukaryotic expression vector pEF1α-HA-σA was successfully constructed,and the recombinant vector could expresse σA in HEK293 T cells. This indicates that the protein can be accurately expressed in HEK293 T cells.
引文
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[8] TOURISOTERO F,MARTNEZCOSTAS J,VAKHARIA V N,et al. Avian reovirus nonstructural protein micro NS forms viroplasmlike inclusions and recruits protein sigma NS to these structures[J].Virology,2004,319(1):94.
[9] XU W,PATRICK M K,HAZELTON P R,et al. Avian reovirus temperature-sensitive mutant ts A12 has a lesion in major core protein sigma A and is defective in assembly[J]. J Virol,2004,78(20):11142-11151.
[10] VZQUEZ-IGLESIAS L,LOSTAL-SEIJO I, MARTNEZCOSTAS J,et al. Different intracellular distribution of avian reovirus core protein sigma A in cells of avian and mammalian origin[J]. Virology,2012,432(2):495-504.
[11] VZQUEZ-IGLESIAS L,LOSTAL-SEIJO I,MARTNEZCOSTAS J,et al. Avian reovirus sigma A localizes to the nucleolus and enters the nucleus by a nonclassical energy-and carrier-independent pathway[J]. J Virol,2009,83(19):10163-10175.
[12]谢丽基,谢芝勋,黄莉,等.禽呼肠孤病毒启动PI3/Akt信号通路的分析[J].畜牧兽医学报,2015,46(9):1613-1619.
[2] SAIF Y M.禽病学[M].高福,索勋,译. 12版.北京:中国农业出版社,2012.
[3]韦平,秦爱建.重要动物病毒分子生物学[M].北京:科学出版社,2008.
[4] DANDR E,BLINT A,KECSKEMTI S,et al. Detection and characterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease[J]. Arch Virol,2013,158(12):2583-2588.
[5] SPANDIDOS D A,GRAHAM A F. Physical and chemical characterization of an avian reovirus[J].J Virol,1976,19(3):968.
[6] XU W,COOMBS K M.Conserved structure/function of the orthoreovirus major core proteins[J].Virus Res,2009,144(1/2):44-57.
[7] SCHNITZER T J,RAMOS T,GOUVEA V. Avian reovirus polypeptides:analysis of intracellular virus-specified products,virions,top component,and cores[J]. J Virol,1982,43(3):1006-1014.
[8] TOURISOTERO F,MARTNEZCOSTAS J,VAKHARIA V N,et al. Avian reovirus nonstructural protein micro NS forms viroplasmlike inclusions and recruits protein sigma NS to these structures[J].Virology,2004,319(1):94.
[9] XU W,PATRICK M K,HAZELTON P R,et al. Avian reovirus temperature-sensitive mutant ts A12 has a lesion in major core protein sigma A and is defective in assembly[J]. J Virol,2004,78(20):11142-11151.
[10] VZQUEZ-IGLESIAS L,LOSTAL-SEIJO I, MARTNEZCOSTAS J,et al. Different intracellular distribution of avian reovirus core protein sigma A in cells of avian and mammalian origin[J]. Virology,2012,432(2):495-504.
[11] VZQUEZ-IGLESIAS L,LOSTAL-SEIJO I,MARTNEZCOSTAS J,et al. Avian reovirus sigma A localizes to the nucleolus and enters the nucleus by a nonclassical energy-and carrier-independent pathway[J]. J Virol,2009,83(19):10163-10175.
[12]谢丽基,谢芝勋,黄莉,等.禽呼肠孤病毒启动PI3/Akt信号通路的分析[J].畜牧兽医学报,2015,46(9):1613-1619.