EGFL7原核表达载体的构建及鉴定

【目的】构建类表皮生长因子域7（EGFL7）原核表达载体，并诱导其表达、纯化及鉴定目的蛋白。【方法】以人肝癌细胞系 HepG2总RNA为模板，PCR扩增 EGFL7基因，克隆至原核表达载体pET‐21a（＋）中，转化大肠杆菌 BL21（DE3），IPTG 诱导表达。 His‐tag 磁珠纯化重组蛋白 EGFL7，SDS‐PAGE 鉴定。【结果】克隆目的基因序列正确，未发生碱基突变；重组表达质粒pET21a（＋）‐TRX‐EGFL7经双酶切鉴定构建正确；表达的重组蛋白相对分子量为80kD ，与预期相符。【结论】成功在大肠杆菌BL21（DE3）中表达并纯化了EGFL7重组蛋白，为后续研究奠定了基础。

Construction and Identification of Prokaryotic Expression Vector of EGFL 7

[Objective]To construct the prokaryotic expression vector of EGFL 7 ,and to induce the expression and purification EGFL7 ,and to identify EGFL7 protein .[Methods]EGFL7 gene was amplified from human hepato‐ma cell HepG2 by PCR and cloned into prokaryotic expression vector pET 21a（＋） ,and then transformed into Esche‐richia coli BL21（DE3） .The expression of EGFL7 was induced by isopropylβ‐D‐1‐thiogalactopyranoside（IPTG） ,and purified by His‐tag magnetic beads ,and identified by SDS‐PAGE .[Results] The sequence of the cloned target gene was correct and had no base mutation .Double enzyme digestion showed that the recombinant plasmid pET 21a （＋）‐TRX‐EGFL7 was constructed correctly .The recombinant protein EGFL7 was expressed with an expected molecular weight of 80kD .[Conclusion]The recombinant protein EGFL7 is successfully expressed and purified in Escherichia coli BL21 （DE3） which sets up the foundation for the following study .