高GC hTwist融合蛋白载体构建及其表达条件优化

Cloning of GC-rich hTwist Preparation,and Charactirization of Fusion Protein

目的构建高GC基因Twist融合蛋白表达载体;高效表达并纯化GST/TWIST融合蛋白,为GST-Pulldown等实验做准备。从而进一步研究其生物学功能。方法以胃癌细胞系SGM7901 cDNA为模板,依据高GC含量基因Twist分子结构特点和高GC含量基因引物设计策略,合成PCR引物。本实验通过分析各种DNA聚合酶不同扩增效率及特异性,选择适合高GC基因扩增的DNA聚合酶,优化反应体系的离子强度,并且结合改良降落PCR等综合策略的实施,比较不同策略下高GC含量基因Twist PCR产物的特异性及效率,尝试高效扩增特异的高GC含量Twist全长编码基因,通过Bam HI和Xho I双酶切位点将Twist全长定向插入pGEX-5X-1载体中,构建原核表达质粒pGEX-5X-1-Twist,并转化E.coli DH5α,筛选富含GC的Twist阳性重组子,限制性内切酶酶切鉴定和DNA序列测序正确后,转化E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,经谷胱甘肽琼脂糖凝胶纯化表达产物,SDS-PAGE和Western blot鉴定。结果通过Platinum pfx DNA聚合酶配合PCRx加强缓冲液的使用及改良降落PCR策略的实施,独立可重复实验证实:我们获得了高效特异的高GC Twist全长编码基因,成功构建原核表达质粒pGEX-5X-1-Twist。诱导表达的GST融合蛋白经SDS-PAGE和Western blot鉴定,检测到分子量正确的特异性蛋白条带,经纯化,得到高纯度的融合蛋白。结论上述策略的实施可以成功完成高GC Twist原核表达载体的构建,并确定GST/TWIST融合蛋白表达的最适条件,获得了高纯度融合蛋白,为进一步研究TWIST蛋白的生物学功能奠定基础。

Objective To construct expression vector of fusion protein GST/TWIST with GC-enriched in Twist,induce expressly,and purify GST/TWIST fusion protein for further studies like GST Pulldown experiment.Methods The GC-rich Twist fragments were amplified by polymerase chain reaction（PCR）with a strategy of ＇multiple factors-employed together＇ and cloned into the expression vector pGEX-5X-1.Recombinant vector of pGEX-5X-1-Twist was identified by restriction enzyme digestion and sequencing.The pGEX-5X-1-Twist recombinant was transformed into E.coli BL21,induced by IPTG.TWIST fusion protein was purified by glutathione beads.The purified product was analyzed by SDS-PAGE and Western blot.Results The GC-rich Twist recombinant pGEX-5X-1-Twist had been successfully constructed.A band corresponding to specific MW protein can be detected by SDS-PAGE after induction.After purification,fusion protein with high purity was harvested.Conclusion The full length gene fragments of human GC-rich Twist were successfully cloned into prokaryotic expression vector.The optimal conditions for inducing the target protein were confirmed and the GST/TWIST fusion protein with high purity was obtained.This study provided a basis for the further research on the biological function of TWIST protein.