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目的构建GST-β-catenin融合蛋白表达载体,并在大肠埃希菌(E.col)中诱导表达。方法从COS-7细胞中提取m RNA,反转录为c DNA。用PCR方法扩增出β-catenin基因全长,通过Bam HI和Sal I酶切位点将其定向插入p GEX-4T-2载体中,构建原核表达质粒p GEX-4T-2-β-catenin,并转化E.coli DH5α,通过限制性内切酶酶切电泳和DNA测序鉴定正确后,转入E.coli BL21中,经异丙基硫代β-D半乳糖苷大量诱导表达,SDS-PAGE电泳和Western blot鉴定。结果酶切电泳及测序结果证明,成功构建了原核表达质粒GST-β-catenin,并用Western blot方法证实了GST-β-catenin融合蛋白的表达。结论成功构建了GST-β-catenin原核表达载体,并证实了其在原核细胞大肠埃希菌中的表达,为进一步纯化β-catenin及研究其结构与功能提供了前提基础。
Objective To construct GST-β-catenin fusion protein expression vector and induce its expression in E.coli. Methods m RNA was extracted from COS-7 cells and transcribed into c DNA. The full-length of β-catenin gene was amplified by PCR and inserted into pGEX-4T-2 vector by Bam HI and Sal I restriction sites to construct prokaryotic expression plasmid pGEX-4T-2-β-catenin , And transformed into E. coli DH5α, identified by restriction enzyme digestion electrophoresis and DNA sequencing, transformed into E. coli BL21, induced by isopropylthio β-D galactoside in large quantities, SDS- PAGE electrophoresis and Western blot identification. Results The results of enzyme digestion electrophoresis and sequencing showed that the prokaryotic expression plasmid GST-β-catenin was successfully constructed and the expression of GST-β-catenin fusion protein was confirmed by Western blot. Conclusion The prokaryotic expression vector of GST-β-catenin was successfully constructed and its expression in prokaryotic Escherichia coli was confirmed. It provides a precondition for the further purification of β-catenin and its structure and function.