荷包猪SLA-2原核表达系的建立及表达研究

Construction of SLA-2 Prokaryotic Expressing System of HeBao Pig and Study on Its Expression

目的:建立荷包猪SLA-2原核表达系并研究SLA-2在pET-32中的表达。方法:设计SLA-2胞外区的表达引物,亚克隆荷包猪SLA-2的胞外区,并转化入原核表达系统pET-32进行表达,SDS-PAGE分析其表达产物的特性。结果:PCR结果显示,SLA-2胞外区亚克隆大小为840bp,酶切鉴定证实成功插入pET-32表达载体,构建重组质粒pET-32a(+)/SLA-2。SDS-PAGE显示,SLA-2基因导入宿主菌后成功表达,蛋白大小为51.4ku,与预期结果相符,蛋白相对表达含量达到了45%。结论:结果表明荷包猪SLA-2在pET-32系统中成功表达,蛋白表达量符合要求,可用于下一步的结构和功能检测。

Objective：To construct the SLA-2 Prokaryotic Expressing System of HeBao Pig,and study on the expression of SLA-2 in pET-32.Method：A pair primer to express the extrocellular domain of SLA-2 was designed and the extrocellular domain of SLA-2 was sub-cloned into pET-32 followed by transforming into Escherichia coli BL21（DE3） and then analysis of the character of expressing products by SDS-PAGE.Result： The PCR results were shown that the length of nucleotides in extracellular domain of SLA-2 was about 840bp which was consistent with the calculated value.In addition,the SLA-2 was proved to be inserted into the pET-32 by cleavage with EcoR Ⅴ and Xho Ⅰ and the constructed expressing vector was named as pET-32a（＋）/SLA-2.By SDS-PAGE,SLA-2 was successfully expressed in Escherichia coli BL21（DE3） and the interest of protein was about 51.4 ku with the 45 percent of relative expressed content of recombinant SLA-2 protein Conclusion： Ii was concluded that the SLA-2 had been expressed in pET-32 Prokaryotic Expressing System and the content of expressed protein satisfied to the need,which would be used to study structure and function of SLA-2.