克隆人RACK1基因,构建原核表达载体pET-30a(+)-RACK1,诱导表达重组蛋白,纯化后Western blot鉴定目的蛋白.根据GeneBank提供的RACK1基因序列及pET-30a(+)载体上的多克隆位点设计引物,以人胎肝cDNA文库为模板,钓取RACK1 cDNA全长序列.将目的片段与原核表达载体pET-30a(+)连接,转入E.coli DH5α感受态细胞,筛选阳性克隆.将测序正确的重组质粒转入E.coli BL21(DE3)细胞,IPTG诱导表达,利用镍柱对表达的蛋白进行纯化,Western blot法检测纯化的蛋白.结果显示,克隆得到的目的基因序列与GeneBank中已报道的序列完全一致,成功构建了pET-30a(+)-RACK1原核表达载体.重组蛋白主要以包涵体形式存在,经镍离子亲和层析柱纯化后,Western blot检测证实纯化的蛋白能与特异性的抗体发生反应.本研究获得了人RACK1蛋白,为深入研究RACK1的功能奠定基础.
Human RACK1 gene was cloned and constructed to pET-30a(+)prokaryotic expression vector.RACK1 protein was expressed and purified and identified by Western blot.A pair of primers was designed according to the digestion sites in pET-30a(+)and the RACK1 gene sequence published on GeneBank.RACK1 cDNA was amplified from a human fetal liver cDNA library and constructed into pET-30a(+)vector.The recombinant expression vector was transformed into the host E.coli DH5α.The positive clone was identified by double enzyme digestion and sequencing analysis.The recombinant expression vector pET-30a(+)-RACK1 was highly expressed in E.coli BL21(DE3)under the induction of IPTG.The fusion protein was analyzed by SDS-PAGE and purified by 6×His-Tagged Protein Purification Kit.The purified RACK1 protein was confirmed by Western blot.The results showed that the RACK1 cDNA sequence we cloned was right.The recombinant expression vector pET-30a(+)-RACK1 was successfully constructed.The fusion protein was expressed as inclusion body, which was purified by affinity chromatography.The result of Western blot indicated that the purified RACK1 protein could bind its specific antibody.The obtained human RACK1 protein laid a good foundation for further study on RACK1.- DOI:10.3969/j.issn.1000-1565.2016.03.012人RACK1基因的克隆及原核表达王向飞,汤静珍,单金帅,刘畅,吴琛(河北大学 生命科学学院,河北 保定 071002)摘 要:克隆人RACK1基因,构建原核表达载体pET-30a(+)-RACK1,诱导表达重组蛋白,纯化后Western blot鉴定目的蛋白.根据GeneBank提供的RACK1基因序列及pET-30a(+)载体上的多克隆位点设计引物,以人胎肝cDNA文库为模板,钓取RACK1 cDNA全长序列.将目的片段与原核表达载体pET-30a(+)连接,转入E.coli DH5α感受态细胞,筛选阳性克隆.将测序正确的重组质粒转入E.coli BL21(DE3)细胞,IPTG诱导表达,利用镍柱对表达的蛋白进行纯化,Western blot法检测纯化的蛋白.结果显示,克隆得到的目的基因序列与GeneBank中已报道的序列完全一致,成功构建了pET-30a(+)-RACK1原核表达载体.重组蛋白主要以包涵体形式存在,经镍离子亲和层析柱纯化后,Western blot检测证实纯化的蛋白能与特异性的抗体发生反应.本研究获得了人RACK1蛋白,为深入研究RACK1的功能奠定基础.关键词:人RACK1;基因克隆;原核表达;蛋白纯化中图分类号:Q78 文献标志码:A 文章编号:1000-1565(2016)03-0293-07Cloning and expression of human RACK1 geneWANG Xiangfei, TANG Jingzhen, SHAN Jinshuai, LIU Chang, WU Chen(College of Life Science, Hebei University, Baoding 071002, China)Abstract:Human RACK1 gene was cloned and constructed to pET-30a(+)prokaryotic expression vector.RACK1 protein was expressed and purified and identified by Western blot.A pair of primers was designed according to the digestion sites in pET-30a(+)and the RACK1 gene sequence published on GeneBank.RACK1 cDNA was amplified from a human fetal liver cDNA library and constructed into pET-30a(+)vector.The recombinant expression vector was transformed into the host E.coli DH5α.The positive clone was identified by double enzyme digestion and sequencing analysis.The recombinant expression vector pET-30a(+)-RACK1 was highly expressed in E.coli BL21(DE3)under the induction of IPTG.The fusion protein was analyzed by SDS-PAGE and purified by 6×His-Tagged Protein Purification Kit.The purified RACK1 protein was confirmed by Western blot.The results showed that the RACK1 cDNA sequence we cloned was right.The recombinant expression vector pET-30a(+)-RACK1 was successfully constructed.The fusion protein was expressed as inclusion body, which was purified by affinity chromatography.The result of Western blot indicated that the purified RACK1 protein could bind its specific antibody.The obtained human RACK1 protein laid a good foundation for further study on RACK1.- 收稿日期:2015-10-23 基金项目:国家自然科学基金资助项目(30800180);河北省自然科学基金资助项目(C2009000191; C2013201112);河北省教育厅优秀青年基金(Y2012024) 第一作者:王向飞(1989-),女,河南洛阳人,河北大学在读硕士研究生.E-mail:18330249598@163.com 通信作者:吴琛(1977-),女,河北保定人,河北大学教授,主要从事肿瘤分子生物学研究.E-mail:dawnwuchen@163.com第3期王向飞等:人RACK1基因的克隆及原核表达
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