›› 2018, Vol. 45 ›› Issue (7): 1791-1797.doi: 10.16431/j.cnki.1671-7236.2018.07.008

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Construction of Chicken RORα Eukaryotic Expression Vector and Its Expression in MSB1 Cell

YOU Zhao1, ZUO Kemeng1, YU Zuhua1, DING Ke1,2, WANG Yaoyao1, LIU Tong1, ZHANG Mengke1, QIU Jingjing1,2   

  1. 1. Key Laboratory of Animal Disease and Public Health of Henan University of Science and Technology, Luoyang 471003, China;
    2. Hongxiang Biological Feed Laboratory of Henan University of Science and Technology, Luoyang 471003, China
  • Received:2017-12-01 Online:2018-07-20 Published:2018-07-20

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Abstract:

In order to construct an eukaryotic expression vector of chicken retinoid acid receptor related orphan receptor alpha (RORα) and detect its expression efficiency in MSB1 cell,the specific primers of RORα gene were designed according to the gene sequence of chicken RORα gene from GenBank (accession No.:NM_001289887.1).Total RNA was extracted from chicken liver tissue,the first strand of cDNA was synthesized by reverse transcription,and the chicken RORα gene fragment was amplified from cDNA,then cloning vector pMD18-T-RORα was constructed.The eukaryotic expression vector pEGFP-N1 and cloning vector pMD18-T-RORα were digested by the restriction enzyme Sal Ⅰ and BamH Ⅰ,the RORα and pEGFP-N1 fragments were recycled,then the RORα gene fragment were connected to pEGFP-N1 to construct the recombinant vector pEGFP-N1-RORα,and the recombinant vector was identified by PCR,double enzyme digestion and squencing.The results showed that the 1 600 bp fragment was amplified from the recombinant eukaryotic expression vector,and two segments length about 4 700 and 1 600 bp were cut using restriction endonuclease Sal Ⅰ and BamH Ⅰ from the recombinant vector pEGFP-N1-RORα.Then the recombinant vector pEGFP-N1-RORα was transfected into the MSB1 cells,the EGFP-positive cells was observed under the fluorescence microscope,and the expression level of RORα in MSB1 cell was evaluated which measured by Real-time quantitative PCR at 48 h after transfection.Compared with control group,the expression level of RORα was significantly increased in pEGFP-N1-RORα transfected MSB1 cell (P<0.05).The result suggested that the eukaryotic expression vector of RORα was successfully constructed,and the recombinant vector could express RORα in MSB1 cell,which laid a foundation to further study the effect of RORα on the biological behavior of MSB1 cell.

Key words: chicken; RORα gene; eukaryotic expression vector; MSB1 cell; expression

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