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

摘要/Abstract

摘要：

为了构建荷包猪SLA-3-HB01基因四聚体前体链原核表达载体，并获得SLA-3-HB01表达蛋白，试验以SLA-3-HB01/pMD18-T为模板进行PCR扩增四聚体前体链SLA-3-HB01-BSP，并克隆至pMD19-T载体中，经NdeⅠ和XhoⅠ双酶切筛选阳性克隆并测序，目的基因连接至表达载体pET-21a（+），转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导表达，SDS-PAGE检测目的蛋白大小及表达情况，提取包涵体并进行检测。结果显示，PCR成功扩增得到SLA-3-HB01-BSP，大小为896 bp左右。酶切鉴定证实，目的基因成功克隆至pMD19-T载体中，插入片段大小为876 bp，阳性克隆经测序后所获序列与原序列一致，并在3'端带有BSP标签序列。酶切鉴定进一步证实成功构建SLA-3-HB01-BSP/pET-21a（+）重组表达载体，经转化及诱导表达，SDS-PAGE检测显示目的蛋白分子质量在33.5 ku左右。包涵体蛋白分子质量约33.5 ku，与菌体中目的蛋白大小一致，经凝胶成像系统UVP扫描分析，包涵体蛋白纯度接近于90%，符合进行相关结构和功能研究的要求。本研究成功构建了荷包猪SLA-3-HB01基因四聚体前体链的pET-21a（+）重组表达系统，并获得了一定纯度的包涵体蛋白。

关键词: 猪白细胞抗原(SLA); 原核构建; 密码子优化; 表达; 包涵体

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

中图分类号:

Q786

高花, 翟晓鑫, 姜平, 甘慧, 张宗辉, 许崇波, 高凤山. 荷包猪SLA-3-HB01基因四聚体前体链的构建及表达[J]. 《中国畜牧兽医》, 2018, 45(10): 2691-2699.

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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