Construction of Eukaryotic Expression Vector of SLA-2-HB01 from Hebao Pigs and Its Expression in PK15 Cells

doi:10.13560/j.cnki.biotech.bull.1985.2018-0695

Abstract

Abstract: This work is to construct a eukaryotic expression vector of SLA-2-HB01 in Hebao pigs and observe its expression in PK15 cells. Firstly,a pair of primers was designed based on the sequence of coding region of SLA-2-HB01 gene,then the gene fragment of coding region of SLA-2-HB01 gene was amplified using SLA-2-HB01-pMD 18-T as template. The positive clones were sequenced after TA cloning and screened by colony PCR. The clones with correct sequences were cleaved and the target genes were recovered to ligate with the eukaryotic expression vector pEGFP N3. The recombinant plasmid SLA-2-HB01/pEGFP N3 was transformed into Escherichia coli firstly and to clone at a high copy way,then a large amount of extracted positive plasmids were transfected into PK15 cells by liposome method. The expression of the SLA-2-HB01 protein was detected by observing the fluorescence intensity of PK15 cells with fluorescence microscopy and by Western blotting. As results,the SLA-2-HB01 with 1 104 bp was successfully amplified,and the recombinant plasmid SLA-2-HB01/pEGFP N3 was constructed successfully. The inserted fragment was proved to be 1 092 bp by enzymatic cleavage. The SLA-2-HB01/pEGFP N3 recombinant plasmid transfected into PK15 cells displayed green fluorescence. Western blotting showed that the molecular weight of the SLA-2-HB01-EGFP fusion protein was 70 kD,which was consistent with the theoretical value. In conclusion,the recombinant plasmid SLA-2-HB01/pEGFP N3 was successfully constructed and expressed in PK15 cells as fusion protein SLA-2-HB01-EGFP,which lays a base for further studying the presentation of peptide epitopes for SLA-2-HB01.

Key words: Hebao pig, PK15 cell, SLA-2, eukaryotic expression vector

ZHAI Xiao-xin, GAO Hua, JIANG Ping, XU Chong-bo, ZHANG Zong-hui, LI Wen-zhe, GAO Feng-shan. Construction of Eukaryotic Expression Vector of SLA-2-HB01 from Hebao Pigs and Its Expression in PK15 Cells[J]. Biotechnology Bulletin, 2018, 34(12): 132-139.

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