牛干扰素-τ在大肠杆菌中的融合表达和鉴定

Fusion Expression of Bovine Interferon-τ Gene in Escherichia coli

牛干扰素-τ(bIFN-τ)是胚胎妊娠识别物质,通过体外重组表达bIFN-τ蛋白用于妊娠识别机理研究和胚胎工程应用具有重要意义。本研究的目的是利用大肠杆菌作进行bIFN-τ蛋白的体外重组表达和鉴定。通过PCR方法扩增得到牛干扰素-τ(bIFN-τ)基因,克隆到pMD18-T载体中。经BamHⅠ和NdeⅠ双酶切回收后连接到原核表达载体pET-28a(+)中,构建了表达质粒(pET-28a-bIFN-τ),然后将其转化大肠杆菌BL21(DE3),并用IPTG进行诱导表达。结果表明:bIFN-τ基因序列插入方向和读码框在pET-28a-bIFN-τ中正确;表达产物分析表明其在大肠杆菌BL21(DE3)中实现了融合表达,表达量占菌体总蛋白的20.68%。优化后的表达条件可使其表达量达到28.84%。重组蛋白主要以包涵体形式存在,经纯化和鉴定,表明表达产物为bIFN-τ蛋白。

The bovine interferon-τ（bIFN-τ） was an important substance in the process of pregnancy recognition,it is greatly significant for the studies on pregnancy recognition mechanism and embryonic bioengineering application that bIFN-τ protein was produced by recombinant expression. The goal of the studies was that the bIFN-τprotein was recombinant expressed in E. coli and identified. Firstly, bIFN-τ gene was obtained by PCR amplification, and then inserted into pMD18-T vector. After pMD18-T inserted bIFN-τ and pET-28a（＋） vectors were digested by BamHⅠ and NdeⅠ, the sequence of bIFN- was linked with prokaryotic expression vector pET-28a（＋）.Thus recombinant fusion expression plasmid pET-28a-bIFN-τ was successfully constructed. Then, pET-28a-bIFN-τ was transformed into E. coli BL21（DE3） and E. coli BL21（DE3） was induced with IPTG for bIFN-τ protein expression. The result showed that the insert direction and reading frame of bIFN-τ gene in pET-28a-bIFN-τ was correct; and E. coli BL21（DE3） expressed bIFN-τ protein with amount of 20.68% of the total bacterial proteins.The expression capacity reached to 28.84% by optimizing expression conditions. The recombinant protein mainly was in inclusion bodies, and the examination confirmed expression product was bIFN-tau protein after purification and identification by western blotting.