Wang Bao-ning, Shi Qiao-fa, Li Hong, Zhang Wei-dong, Zhuang Yong-hua, Yang Jing, Jiang Zhong-hua, Li Ming-yuan
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2006 May;37(3):361-4.
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.
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.
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 degrees C or 37 degrees C for 18 hours. The results of SDS-PAGE 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.
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.
克隆人乳头瘤病毒16型（HPV - 16）E4基因，构建原核表达工程菌，探讨其表达条件及表达产物特性。
从经Real - PCR确诊为HPV - 16阳性的临床宫颈病变样本细胞提取物中，通过PCR克隆完整的E4基因。将E4 DNA片段插入pET32a(+)构建原核表达质粒pET32/E4，然后将表达质粒转入感受态大肠杆菌BL21(DE3)。用Bgl II和Hind III酶切鉴定重组DNA，随后测序。用不同浓度IPTG在不同温度下诱导重组菌BL21/E4。通过SDS - PAGE和Gel - Pro Analyzer 4检测和分析表达的蛋白。将BL21/E4表达蛋白的His标签与单克隆抗体杂交。
PCR克隆的E4基因约342 bp。比对结果显示该E4基因与HVP - 16 DNA序列有99%的同源性，克隆的E4基因表达框与HVP - 16东亚株相同。与GenBank中其他HPV - 16株相比，E4基因同源性在97%以上。pET32/E4能在BL21中表达重组E4（rE4）。当BL21/E4在28℃或37℃用0.1 mmol/L IPTG诱导18小时时，表达量最高，分别占总细菌蛋白的12.2%或12.8%。SDS - PAGE和Western blot结果显示rE4主要以包涵体形式表达，并与His标签融合（rE4/His），该融合蛋白可溶，分子量约为34 kDa。
成功从HPV - 16中克隆E4基因，构建了能有效表达与His标签融合的rE4蛋白（rE4/His）的原核表达大肠杆菌BL21/E4。该融合蛋白能与识别His标签的单克隆抗体反应，便于通过亲和层析纯化。上述研究结果为制备高纯度E4蛋白及开展相关研究奠定了良好基础。
