关键词: 荷包猪; 猪白细胞抗原1; 四聚体; 可生物素酰化序列

Abstract:

To construct tetramer precursor chain of swine lymphocyte antigen 1 (SLA-1) heavy chain in Hebao pig and study its protein expression in the pET-21a (+) vector,the SLA-1 complete genome sequence was referenced with the characteristics of the expression vector,and a pair of primers was designed to integrate the BirA substrate peptide (BSP) sequence at the C-terminus of the SLA-1-HB01 and the SLA-1-HB01-BSP was amplified by PCR. Then,the products were cloned into the pEASY T1 vector and the positive clones of SLA-1-HB01-BSP/pEASY T1 was selected. After the double digestion,the interest of the gene in positive clone was further ligated into the pET-21a (+) expression vector and transformed into E.coli BL21 to construct the recombinant strain of pET-21a (+)/SLA-1-HB01-BSP. After induction with IPTG,the target protein were detected by SDS-PAGE. Finally,the inclusion body of the SLA-1-HB01-BSP was isolated and detected to evaluate its purity. The PCR results showed that the length of SLA-1-HB01-BSP was about 898 bp,which was consistent with the theoretical value. The amplified fragment was successfully cloned into pEASY T1 vector, and the positive clones were identified by Nde Ⅰ and Xho Ⅰ digestion. The size of inserted fragment was 876 bp. The interest of gene was also inserted into pET-21a (+) and transformed into E.coil BL21 successfully. After induction,SDS-PAGE detection results showed that the target protein was 31.4 ku. Further detection showed that the target protein was mainly expressed as inclusion bodies,and the purity of the protein was about 80%. In this study,the recombinant tetramer precursor of SLA-1-HB01 heavy chain was constructed in pET-21a (+) expression line successfully, which would lay a foundation to detect the tetramer of SLA class Ⅰ molecular.

Key words: Hebao pig; SLA-1; tetramer; BirA substrate peptide