人乳头状瘤病毒86 E7重组蛋白的高效表达、纯化和表征

Expression, purification and characterization of recombinant human papillomavirus 86 E7

关键词:  人乳头状瘤病毒  E7蛋白  纯化  表征  结构

Key words:  human papillomavirus (HPV)  E7  purification  characterization  structure

摘要:

［目的］基因工程法构建谷胱甘肽S-转移酶(GST)-人乳头状瘤病毒（Human papillomavirus）HPV86 E7融合蛋白表达质粒，高效表达、纯化、鉴定蛋白质和分析蛋白质的存在形式。［方法］GST-HPV86 E7融合蛋白基因与pGEX-6P-1载体连接成重组表达质粒pGEX-6P-1-GST-HPV86 E7，重组质粒转入BL21大肠杆菌,经IPTG诱导表达融合蛋白GST-HPV86 E7，柱上切除GST标签，Glutathione Sepharose 4B亲和层析纯化蛋白,SDS-PAGE、MALDI-TOF和色谱质谱联用系统分别用于蛋白质纯度分析、分子量测定和蛋白鉴定；非变性电泳和分子筛排阻色谱用于蛋白质四级结构的分析。［结果］HPV86 E7大肠杆菌中正确表达,基质辅助激光解析飞行时间质谱测得的HPV86 E7精确分子量为(11319.76±5.66) Da。反相高压液相色谱-电喷雾串联质谱鉴定的匹配肽段有11个肽段，氨基酸的覆盖率为90%。非变性PAGE和凝胶过滤层析实验观察到了HPV86 E7的单聚体、二聚体及多聚体。［结论］重组质粒pGEX-6P-1-GST-HPV86 E7成功构建,诱导后高效表达GST-HPV86 E7融合蛋白，HPV86 E7得到纯化,质谱分析证实表达蛋白为86E7蛋白，以多聚体形式存在。

Abstract:

［Objective］To construct the plasmid of GST-HPV86 E7 fusion protein and to overexpress the recombinant GST-HPV86 E7, to purify and identify the HPV86 E7,and to analyze the existence state HPV86 E7 in aqueous solution.［Methods］GST-HPV86 E7 fusion gene is constructed first and then inserted into the pGEX-6P-1 vector. The recombinant plasmid is transferred into BL21 (DE3) and the fusion protein is overexpressed in E. Coli with the induction of IPTG. The GST tag is removed by using the PreScission protease and the HPV86 E7 is purified to homogeneity through two Glutathione Sepharose 4B columns in tandem. SDS-PAGE, MALDI-TOF and ESI-LC-MS/MS are used to analyze the purity, the molecular mass and the identity of the HPV86 E7. Native PAGE and size exclusion chromatograph are used to measure the quaternary structure of the purified protein.［Results］The protein of HPV86 E7 was correctly expressed in E. Coli. The molecular mass of purified HPV86 E7 is (11319.8±5.66)Da by MALDI-TOF mass spectrometry analysis. 11 peptides, produced by the tryptic and Asp-N digestions, that occupy 90% of sequence coverage of HPV86 E7, were identified by the HPLC-ESI-MS/MS analysis. The monomer, dimer and multimer of HPV86 E7 were observed by native PAGE and gel filtration chromatography experiments.［Conclusions］The pGEX-6P-1-GST-HPV86 E7 recombinant plasmid is successfully constructed and the GST-HPV86 E7 is overexpressed with high solubility. HPV86 E7 is obtained in high purity and is identified by mass spectrometric analysis. The purified HPV86 E7 exists as multimer in aqueous solution.