كلونينگ ژن كدكننده آنتي ژن سطحي ويروس هپاتيت B در اشرشياكولي

Cloning of Hepatitis B Virus Surface Antigen(HbsAg) Encoded Gene in E.Coli

Abstract Background & Aim: Hepatitis B virus(HBV) infection is endemic worldwide. It is estimated every year more than 350 million people become infected with HBV(new cases) worldwide. Unfortunately, there are no satisfactory drugs to cure HBV and related diseases and the only way to control it is through vaccination. Measurements of HBV DNA levels are routinely used to identify infectious chronic carriers and to predict and monitor the efficacies of antiviral treatment regimens. The quantification of HBV DNA in clinical specimens has significantly improved with the introduction of real-time PCR into routine diagnostic laboratories. The aim of this research is to study cloning and identify hepatitis B virus surface antigen(HbsAg) encoded gene as external standard. Material and Methods: A descriptive study was done on hepatitis B virus sufrace antigen encoded gene. Genomic DNA was extracted from HbsAg positive serum sample and amplified by PCR. The amplified segments were cloned in pTZ57R plasmid vector. After purification the PCR product was cloned in plasmid vector and transformed into susceptible E.coli(TGl strain) cell. The bacteria containing the new combination plasmid was than evaluated for antibiotic resistance, PCR, enzyme cleavage and sequencing. The mean of colony numbers that grew in plates with and without ampicilin and also statistical analysis, T-student test was used. Results: After extraction of viral DNA, Sgene was amplified by PCR; 175 bp fragment was generated. S gene was than cloned by PT2 57R plasmid. The new plasmid was extracted and was confirmed by enzyme cleavage and PCR. For final confirmation it underwent sequencing. Conclusion: According to the results the 175 bP fragment of s gene was cloned into PT257R Plasmid. It can be used as external standard for real time PCR or as a positive control in laboratory.