[Analysis of protein expression feature and construction of procaryotic expression system for human papillomavirus type 16 (HPV-16) E4 gene].
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.


CONCLUSIONS
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.
Wang
Bao-ning
BN
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Shi
Qiao-fa
QF
Li
Hong
H
Zhang
Wei-dong
WD
Zhuang
Yong-hua
YH
Yang
Jing
J
Jiang
Zhong-hua
ZH
Li
Ming-yuan
MY
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D000914:Antibodies, Viral / Q000096:biosynthesis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D015967:Gene Expression Regulation, Viral; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000235:genetics; D030361:Papillomavirus Infections / Q000821:virology*; D010957:Plasmids / Q000235:genetics; D011387:Prokaryotic Cells / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D002575:Uterine Cervicitis / Q000821:virology
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; C069119:oncogene protein E4, Human papillomavirus type 16