ABSTRACT
Objective To construct recombinant plasmids containing HPV18E7 gene ,and explore the optimization condition of its expression in Escherichia coli .Methods The genomic DNA extracted from HeLa cell line which served as a template to the HPV18 E7 gene was amplified using PCR method ;and the amplified product of HPV18E7 gene was connected to the pET-32a(+ ) vector ,which composed the pET-32a(+ )-HPV18E7 recombinant plasmid ;the positive recombinant plasmids were transformed into BL21-DE3-pLysS competent cells and the optimized expression condition was explored in order to obtain a large amount of HPV18E7 oncogenic protein .Results The fragment length of PCR products of HeLa cell genomic DNA was consistent with that of HPV18 E7 gene .In LB medium ,the expression level of the target protein was not high under such conditions as different concentra-tion of IPTG and lactose ,different temperatures and different induction starting amount .Therefore the ZYM-5052 auto-induction medium was tried in this experiment ,and the expression amount of the fusion protein was much higher than that induced with IPTG and lactose .Conclusion The amount of HPV18E7 fusion protein in ZYM-5052 automatic induction medium is much higher than that induced with IPTG and lactose .
ABSTRACT
BACKGROUND: Lung cancer is the leading cause of cancer deaths in the world. Human papillomavirus (HPV) infection and E6 oncoprotein expression are known risk factors for the development of non-small cell lung cancer (NSCLC). This study was performed to evaluate the prevalence of HPV 16/18 E6 oncoprotein expression in patients with NSCLC. METHODS: Immunohistochemical stains of the HPV 16/18 E6 oncoprotein were performed in tumor tissues from 68 patients with NSCLC who underwent curative surgery from March 2006 to November 2008. RESULTS: The E6 oncoprotein was expressed in 29.4%of patients with NSCLC and a statistical analysis revealed that E6 oncoprotein expression was significantly higher in females (p=0.028), never smokers (p=0.045), and patients with adenocarcinoma (p=0.022) than that in other patients. CONCLUSION: The E6 oncoprotein was expressed in 29.4% of patients with NSCLC. Further studies detecting HPV infection and E6 oncoprotein expression in never smoking patients with NSCLC are needed.
Subject(s)
Female , Humans , Adenocarcinoma , Carcinoma, Non-Small-Cell Lung , Coloring Agents , Lung Neoplasms , Oncogene Proteins, Viral , Prevalence , Risk Factors , Smoke , SmokingABSTRACT
Objective To construct a recombinant plasmid for expressing the HPV18 E2 gene and develop some materials for HPV18 related disease.Methods The E2 gene of HPV18 was amplified by PCR from PBR322-HPV18 and cloned into PIRES2-EGFP.The sequence of cloned HPV18E2 was confirmed by restriction analysis and DNA sequencing,then transfected into HeLa cells.E2 gene was detected by RT-PCR.Results HPV18 E2 gene fragment was amplified by PCR and ligated to PIRES2-EGFP,then transfected into HeLa cells successfully.The expression of green fluorescence cells was observed by fluorescence microscopy.Specific band could be seen in transfected cells by RT-PCR.Conclusion Recombinant plasmid PIRES2-EGFP is effectively expressed after being transfected into HeLa cells in vitro.