高GC hTwist表达载体构建及其表达条件优化

 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，成功构建了原核表达质粒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，经纯化后，得到了高纯度的融合蛋白。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了基础。

Cloning of GC- rich hTwist and induced GST/TWIST Expression

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.