Biotechnology Bulletin ›› 2021, Vol. 37 ›› Issue (10): 143-151.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1401

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Construction of a Eukaryotic Expression Vector of SLA-2 Gene from Yantai Black Pigs and Its Expression

HU Xiao(), WANG Bao-bao, DOU Shao-hua, JIANG Nan, FU Chang-zhen, JIN Hang, GAO Feng-shan()   

  1. College of Life Science and Technology,Dalian University,Dalian 116622
  • Received:2020-11-17 Online:2021-10-26 Published:2021-11-12
  • Contact: GAO Feng-shan E-mail:eeeeyouxiang@163.com;gfsh0626@126.com

Abstract:

In order to construct the recombinant eukaryotic expression vector SLA-2-YT/pCDH from a Yantai black pig’s SLA-2 gene(SLA-2-YT)and to express it in eukaryotic cells,a pair of primers were designed according to SLA-2-YT coding region gene sequence,and the SLA-2-YT coding region gene fragment was amplified by PCR using SLA-2-YT/pMD18-T whole-gene cloning expression vector. The restriction enzyme Xba I and Not I were added to the 5'end of the forward and lower primers,respectively. The target gene was firstly cloned into pMD 19-T Simple Vector TA and was ligated to the pCDH-CMV-MCS-EF1-Puro(pCDH)eukaryotic expression vector. The recombinant plasmids were transformed into Escherichia coli Stbl 3 competent cells. The recombinant plasmids were extracted from the expanded culturing monoclonal strain and the sequences were verified by double enzyme digestion and sequencing. A large quantity of endotoxin-free plasmids was extracted from the recombinant eukaryotic expression vector with correct inserting sequence,and then they were transfected into sT2 cells through lentivirus packaging and infection. The expression of SLA-2-YT gene in sT2 cells was detected by Western Blotting. The results showed that the recombinant eukaryotic expression vector SLA-2-YT/pCDH was successfully constructed. After packaging by lentivirus,the recombinant plasmids infected sT2 cells followed by puromycin screening,and then a positive cell clone was successfully obtained. Western Blotting detection showed that SLA-2-YT/pCDH was predominantly expressed in sT2 cells with the molecular weight of 45 kD,which was consistent with the theoretical designing value. In this experiment,the SLA-2-YT coding region gene was successfully constructed into the eukaryotic expression vector,and predominantly expressed in sT2 cells,which will supply good materials for studying CTL epitoes presented by SLA-2-YT in future.

Key words: Yantai black pig, SLA-2 gene, eukaryotic expression vector, lentivirus packaging, transfection