人源S100A1蛋白的表达纯化与鉴定

EXPRESSION,PURIFICATION,AND IDENTIFICATION OF HUMAN S100A1 PROTEIN

目的构建pBV220-S100A1表达载体,表达并纯化人源S100A1蛋白,为进一步研究人源S100A1蛋白相关的生物学功能及应用于临床诊断奠定基础。方法采用全基因合成法合成人源S100A1基因;通过GeneBank查询人源S100A1蛋白的氨基酸序列,进行序列优化设计,同时引入EcorI和BamHI酶切位点,构建原核表达载体pBV220-S100A1,热应激诱导其在大肠杆菌(E.coli DH5α)中表达,采用离子交换层析法纯化人源S100A1蛋白,最后采用Transwell迁移实验检测所纯化蛋白的生物活性。结果酶切鉴定和测序分析显示,人源S100A1的基因被成功插入pBV220多克隆位点;S100A1的基因片段在pBV220中的插入位点、碱基序列及读码框和终止子的次序完全正确;S100A1的基因序列位于表达载体pBV220的序列下游。经诱导表达和纯化,最终获得了人源S100A1蛋白,且经迁移实验证明,该纯化蛋白具有较高的生物活性。结论成功建立了人源S100A1蛋白表达载体,表达菌株经诱导表达和纯化,获得了高纯度的蛋白。

Objective To construct a pBV220-S100A1 expression system for expressing and purifying human S100A1 protein,in order to provide an efficient and convenient tool for further investigating the biological function of S100A1 protein and application to clinical diagnosis.Methods Human S100A1 genes were synthesized by whole gene synthetic process and its amino acid sequence was obtained from genebank.The sequence was optimized by inserting the suitable restriction enzyme sites including Ecor I and BamH I.To construct the prokaryotic pBV220-S100A1 expression vector,the fragment by PCR was inserted into pBV220.And the recombinant protein was over-expressed in Escherichia coli DH5α by heat shock.Human S100A1 protein was purified by anion-exchange chromatography.Finally,the biological activity of purified protein was detected with Transwell migration assay.Results The cDNA sequence of S100A1 was correctly inserted into the multicloning site of pBV220 by restriction enzyme digesting and DNA sequencing.Sequence analysis showed that the inserting site,base sequence,reading frame,and stop codon of S100A1 in pBV220-S100A1 vector were completely correct.Human S100A1 protein was successfully purified from Escherichia coli DH5α by anion-exchange chromatography,and the purified protein had a high biological activity.Conclusion Prokaryotic pBV220-S100A1 expression vector was successfully constructed.And the highly purified protein were gained after expression,and purification.