合成基因IL-6在大肠杆菌中表达及融合蛋白鉴定分析  

Analysis of fusion protein identification and expression of synthetic gene IL-6 in E.coli

作　　者：唐维政 孙鸿高 陈少文 汪凡 朱中元 Tang Weizheng;Sun Honggao;Chen Shaowen;Wang Fan;Zhu Zhongyuan(Department of Clinical Laboratory,The Second Affiliated Hospital of Hainan Medical University,Haikou 570311,Hainan,China)

机构地区：[1]海南医学院第二附属医院检验科,海南海口570311

出　　处：《贵州医药》2020年第5期682-684,共3页Guizhou Medical Journal

基　　金：海南省卫生计生行业科研项目(项目编号:19A200122)。

摘　　要：目的合成IL-6的编码基因,并在大肠杆菌中表达,获得纯化的融合蛋白IL-6。方法采用基于PAS(PCR-based accurate synthesis)的方法,设计全长拼接引物,合成基因IL-6,并将其插入载体pCzn1,获得的重组质粒pCzn1-IL-6,化学法将其转化入大肠杆菌TOP10进行克隆。将克隆得到的阳性克隆子转化入大肠杆菌Arctic Express后用IPTG诱导剂诱导融合蛋白的表达,SDS-PAGE和Western Blot鉴定蛋白表达效果。结果经测序和双酶切验证,正确构建了重组质粒pCzn1-IL-6,重组质粒成功转入表达菌株,IL-6原核蛋白在表达菌株中以包涵体和可溶性形式稳定表达,通过Ni柱亲和纯化获得纯化的目标蛋白。结论 IL-6原核蛋白在大肠杆菌中成功表达,为进一步的应用研究打下基础。Objective To synthesize the coding gene of IL-6 and express it in E.Coli,and the purified fusion protein IL-6 was obtained.Methods Using the method based on PAS(PCR based accurate synthesis),it designed a full-length splicing primer,synthesized the gene IL-6.Recombinant plasmids(pCzn1-IL-6)were constructed by inserted the gene IL-6 into the vector plasmids pCzn1 and then were cloned by being transformed into E.coli TOP10.Subsequently,the cloned positive recombinant plasmid pCzn1-IL-6 was transformed into E.coli Arctic Express,the fusion proteins of IL-6 were induced with IPTG inducer,and identified by SDS-PAGE and western blot(WB).Results Constructed the recombinant plasmid pCzn1-IL-6 which verified by sequencing and double enzyme digestion.The recombinant plasmid was transformed into expression strain E.coli.The prokaryotic protein of IL-6 existed as inclusion bodies and soluble in E.coli.The Purified protein of IL-6 was obtained by affinity purification on Ni column.Conclusion IL-6 prokaryotic protein was successfully expressed in E.coli,which laid a basis for further application research.

关 键 词：IL-6 合成基因 原核表达 融合蛋白 

分 类 号：R34[医药卫生—基础医学] R37