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 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.
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.