荷包猪SLA-3-HB01基因四聚体前体链的构建及表达

doi:10.16431/j.cnki.1671-7236.2018.10.005

Abstract

Abstract:

In order to construct the prokaryotic expression vector of tetramer precursor chain of SLA-3-HB01 gene in Hebao pig and express the SLA-3-HB01 protein,using SLA-3-HB01/pMD18-T as a template,one pair of primers was designed to amplify the tetramer precursor chain named as SLA-3-HB01-BSP by PCR.The SLA-3-HB01-BSP was cloned into pMD19-T simple vector,the positive clones were double digested by NdeⅠ and XhoⅠ followed by sequencing.The target gene was further ligated into the expression vector pET-21a (+) and transformed into Escherichia coli BL21(DE3).After induction with IPTG,the size of expressed protein was detected by SDS-PAGE,and then inclusion bodies of SLA-3-HB01-BSP protein were extracted and detected.The results showed that SLA-3-HB01-BSP was amplified successfully,the size of product was about 896 bp.The double digestion with NdeⅠ and XhoⅠ demonstrated that the target gene was successfully cloned into pMD19-T simple vector with the inserted size of 876 bp.After sequencing and analyzing,it was proved that the target gene was consistent with the primary gene in sequence and a BSP tag was identified on the 3' end of the target gene.The recombinant expression vector SLA-3-HB01-BSP/pET-21a(+) was successfully constructed.SDS-PAGE detection result showed that the molecular weight of the target protein was about 33.5 ku.The molecular weight of inclusion body was about 33.5 ku,which was consistent with that of the target protein in mycoproteins.Scanning by UVP in Gel Imaging system,it was shown that the purity of inclusion body of protein was about 90%,which was suited for the requirements to study the related structure and function of the protein.In this study,the recombinant expression vector pET-21a (+) for SLA-3-HB01 tetramer precursor chain was successfully constructed,and inclusion body of protein with a certain purity was obtained.

Key words: SLA; prokaryotic construction; codon optimization; expression; inclusion body

CLC Number:

Q786

GAO Hua, ZHAI Xiaoxin, JIANG Ping, GAN Hui, ZHANG Zonghui, XU Chongbo, GAO Fengshan. Construction and Expression of Tetramer Precursor Chain of SLA-3-HB01 Gene in Hebao Pig[J]. , 2018, 45(10): 2691-2699.

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Construction and Expression of Tetramer Precursor Chain of SLA-3-HB01 Gene in Hebao Pig

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