HAI-1基因不同功能域的原核表达载体构建及融合蛋白表达

目的:分段克隆1型肝细胞生长因子激活剂抑制因子(HAI-1)基因的功能域并在大肠杆菌中表达,为进一步研究HAI-1的生物学功能打下基础。方法:针对HAI-1的不同功能域设计相应的5对引物,分别扩增KD1、KD1 LDLR、KD2、LDLR KD2和KD1 LDLR KD2片段。将PCR产物克隆至pGEM-TEasy载体中并经测序鉴定。将5个cDNA片段亚克隆至具有6个组氨酸标签(His-Tag)的原核表达载体pIVEX2.3-MCS中。以上述表达不同功能域的载体转化大肠杆菌BL21(DE3)菌株,用IPTG诱导融合蛋白的表达,表达产物以Westernblot进行鉴定,并进一步通过镍亲和层析柱纯化His-Tag标记的KD1 LDLR KD2融合蛋白。结果:成功地构建了HAI-1各功能域基因片段的原核表达载体。Westernblot分析证实,在大肠杆菌BL21(DE3)中分段表达了5种HAI-1不同功能域的His-Tag融合蛋白,并通过亲和层析法纯化到了相对分子质量为22200的KD1 LDLR KD2融合蛋白。结论:重组质粒pIVEX2.3-MCS/HAI-1能在大肠杆菌BL21(DE3)中表达,纯化的融合蛋白可用于研究HAI-1不同功能域的生物学作用。

Construction of prokaryotic expression vectors of HAI-1 with different domains and expression of the fusion proteins

AIM: To construct prokaryotic expression vectors for different domains of hepatocyte growth factor activator inhibitor type 1(HAI-1) gene and express the fusion proteins for further investigation into their biological functions. METHODS: Five pairs of primers were designed to amplify different cDNA fragments of HAI-1 (KD1, KD1+LDLR, KD2, LDLR+KD2 and KD1+LDLR+KD2). The PCR products were cloned into pGEM-T Easy vectors and sequenced. The cDNA fragments were subcloned into pIVEX2.3-MCS, a prokaryotic expression vector with 6xhis tag and transformed into E.coli BL21 (DE3). The expression of histidine-tagged fusion proteins were induced with IPTG and confirmed by Western blot. The histidine-tagged fusion protein KD1+LDLR+KD2 was purified through immobilized Ni(2+) absorption chromatographic column. RESULTS: Different domains of HAI-1 with 6 x his tag were successfully expressed in E.coli BL21 (DE3). The expressed KD1+LDLR+KD2 domain with the relative molecular mass size of 22,200 was purified specifically through affinity chromatographic column and confirmed by Western blot. CONCLUSION: Recombinant plasmid pIVEX2.3-MCS/HAI-1 can be expressed in E.coli BL21(DE3) and the purified fusion protein KD1+LDLR+KD2 can be used for further research on biological functions of different domains of HAI-1.