[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according ...[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according to the gene sequence of ARV S2 gene in GenBank(accession number KF741763.1).With pMD18-T-σA recombinant vector as the template,the specific sequence ofσA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant plasmid.The cloning vector pMD18-T-σA and eukaryotic expression plasmid pEF1α-HA were double digested by restriction enzymes Kpn I and Not I.The purifiedσA gene was connected with pEF1α-HA to construct eukaryotic expression plasmid pEF1α-HA-σA.After colony PCR,double enzyme digestion and sequencing,the recombinant plasmid pEF1α-HA-σA was tansfected into HEK293T cells.The proteins were collected at 24 h after tansfection and verified by Western-blot.[Result]The ARVσA gene was successfully cloned in the test.The eukaryotic expression plasmid pEF1α-HA-σA was constructed,which could be expressed in HEK293T cells.[Conclusion]The protein could be accurately expressed in HEK293T cells.展开更多
基金Supported by Guangxi Science Base and Talents Special Program(AD17195083)Guangxi Science Great Special Program(AA17204057)+4 种基金National Natural Science Foundation of China(3166071531160512)Science and Technology Project of Guangxi Province(2018GXNSFAA138106)Guangxi Bagui Scholars Program Foundation(2019-79)National Ten-Thousand Talents Program of China(W02060083).
文摘[Objective]The paper was to construct eukaryotic expression vector of Avian reovirus(ARV)σA gene and expressσA protein accurately in HEK293T cells.[Method]The specific primers of ARVσA gene were designed according to the gene sequence of ARV S2 gene in GenBank(accession number KF741763.1).With pMD18-T-σA recombinant vector as the template,the specific sequence ofσA gene was amplified by PCR and cloned into pMD18-T vector to construct recombinant plasmid.The cloning vector pMD18-T-σA and eukaryotic expression plasmid pEF1α-HA were double digested by restriction enzymes Kpn I and Not I.The purifiedσA gene was connected with pEF1α-HA to construct eukaryotic expression plasmid pEF1α-HA-σA.After colony PCR,double enzyme digestion and sequencing,the recombinant plasmid pEF1α-HA-σA was tansfected into HEK293T cells.The proteins were collected at 24 h after tansfection and verified by Western-blot.[Result]The ARVσA gene was successfully cloned in the test.The eukaryotic expression plasmid pEF1α-HA-σA was constructed,which could be expressed in HEK293T cells.[Conclusion]The protein could be accurately expressed in HEK293T cells.
