摘要
目的克隆人乳头瘤病毒-16型(HPV-16)E4基因(E4),构建E4蛋白表达重组工程菌,探索表达条件和鉴定表达产物特性。方法以临床HPV-16阳性宫颈炎患者上皮细胞提取物为模板,PCR扩增E4完整基因,并定向克隆到pET32a(+)原核表达质粒中,经双酶切和测序鉴定后转入大肠杆菌BL-21(DE3),获得工程菌(BL-21/E4)。经IPTG诱导,表达蛋白用SDS-PAGE和Westernblot鉴定。结果克隆到完整E4基因为342bp,与HPV-16东亚型的同源性为99%,与其他HPV16型的同源性高于97%。在28℃和37℃,0.1mmol/L的IPTG诱导18h时,重组蛋白(rE4)表达量分别占菌体总蛋白的12.2%和12.8%;SDS-PAGE和Westernblot证明rE4蛋白与表达载体的标签蛋白形成相对分子质量约34×103的可溶性融合蛋白(rE4/His)。结论成功构建了表达带标签的融合重组蛋白(rE4/His)的重组大肠杆菌BL-21/E4表达系统,这为高纯度HPV-16E4蛋白的制备和相关研究奠定了基础。
Objective To clone the E4 gene （E4） from human papillomavirus type 16（HPV-16）, construct the engineering bacteria of prokaryotic, expression, and explore the expression conditions and the characters of expression product. Methods The complete E4 gene was cloned by PCR from the sample cell extract of clinical cervical disease that was the positive HPV-16 confirmed by Real-PCR. The E4 DNA fragment was inserted into the pET32a（＋） to construct a prokaryotic expression plasmid, called as pET32/E4. Then the expression plasmids were transferred into competent E. coli BL21 （DE3）. Recombinant DNA was identified by Bgl Ⅱ and Hind Ⅲ digestion, and then sequencing. The recombine bacterium, BL21/E4, was induced with different IPTG concentrations at different temperatures. The expressed proteins were checked and analyzed by SDS-PAGE and Gel-Pro Analyzer 4. His-tag of BL21/E4 expression protein was hybridized to McAb. Results The E4 gene cloned by PCR was about 342 bp. The blasted result showed that the E4 gene had 99% homology .of HVP-16 DNA sequence, the cloned E4 gene expression frame was the same as HVP-16 East Asia strain＇s. Compared with other HPV-16 strains in GenBank, the homology of E4 gene was above 97%. pET32/E4 could express recombinant E4 （rE4） in BL21. The highest expression, which was 12.2% or 12.8% of total bacterial proteins respectively, was gotten when BL21/ E4 was induced by 0. 1 mmol/L IPTG at 28 ℃ or 37 ℃ for 18 hours. The results of SDSPAGE and Western blot showed the rE4 was expressed mainly to form the inclusion body, and to fuse with his-tag （rE4/His）, that was soluble and had a molecular weight as about 34 KDa. Conclusion We cloned successfully the E4 gene from HPV-16 and constructed the prokaryotic expression E. coli BL21/E4, which could expression rE4 protein fused with his-tag （rE4/His）, effectively. The fused protein could react to McAb recognizing His-tag, which was convenience purified by affinity chromatography. The above research results built a good foundation for preparing the high grade of purity E4 protein and developing the relative study.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第3期361-364,共4页
Journal of Sichuan University(Medical Sciences)
关键词
HPV-16
E4基因
原核表达
Human papillomavirus typel6 E4 gene Prokaryotic expression
