Production of human papilloma virus type 16 E6 oncoprotein as a recombinant protein in eukaryotic cells

Cervical cancer is one of the most important and widespread cancer which affects women. There are several causes of cervical cancer; among them HPV types 16 and 18 are the most prominent ones which are recurrent and persistent infections. These genotypes are currently about 70% of cervical cancer causes in developing countries. Due to the importance of these viruses in cervical cancer, we pioneered the production of Human Papilloma Virus type16 E6 oncoprotein as a recombinant protein in order to develop a vaccine. Two HPV oncoproteins, E6 and E7, are consistently expressed in HPV-associated cancer cells and are responsible for malignant transformation. These oncogenic proteins represent ideal target antigens for developing vaccine and immunotherapeutic strategies against HPV-associated neoplasm. In the present study, the cloned E6-oncoprotein of HPV16 in pTZ57R/TE6 vector was used to produce professional expression vector. The target gene was subcloned in a eukaryotic expression vector. The pcDNA3-E6 vector was propagated in E.coli strain DH5 alpha and transfected into CHO cells 72 hours posttransfection. The transfected cells were harvested; mRNA detection and the interest protein production were confirmed by western blot analysis using specific anti E6 monoclonal antibody. HPV16-E6 target protein recognized by specific antibody could be an appropriate form of protein, which can be used for further studies. Due to potential effect of this protein, its DNA construction can be used for DNA vaccine in future studies

Comparison of enzyme immunoassay, immunochromatography, and RNA-polyacrylamide-gel electrophoresis for diagnosis of rotavirus infection in children with acute gastroenteritis

Human rotavirus is a major etiologic agent for infantile diarrhea worldwide. It is responsible for up to 3.3 million deaths per year in children in developing countries. Various rapid and sensitive techniques have been developed to readily diagnose rotavirus gastroenteritis. In the present study, we compared the sensitivity and specificity of immunochromatography and RNA-polyacrylamide-gel electrophoresis [PAGE] methods with enzyme immunoassay [EIA] for diagnosis of group A rotavirus infection in 200 stool samples from children younger than 5 years old with acute gastroenteritis. Rotavirus was detected in 57 [28.5%] samples by EIA, 52 [26%] samples by ICG and 52 [26%] samples by RNA-PAGE. There was no significant difference between the three methods [P=0.8] nor between EIA and ICG [P=0.57] and EIA and RNA-PAGE [P=0.57]. Furthermore, in comparing these methods with age variables, the present study found that the sensitivity and specificity of ICG and RNA-PAGE compared with EIA were 87.7%, 98.6% and 91.2%, and 100%, respectively [P>0.05]. Results of the present study demonstrate that the sensitivity and specificity rates for ICG and RNA-PAGE were as high as EIA. It seems that all the three methods are reliable and suitable for detection of group A rotavirus infection in children affected by enteric diseases