E.COLI نوتركيب به همراه برچسب هيستيديتي با ساختار فيزيولوژيك طبيعي ، بدنبال كلونينگ و بيان آن در HBEAG تخليص و ارزيابي خواص

Hepatitis B virus (HBV) is considered a global health problem which affects 360 millions of people worldwide and about two millions in Iran. Among HBV antigens, the physiological function of HBeAg is still unresolved. In this respect, its presence in positive patients is regarded as a marker of viral replication and it may have roles in pathogenesis and persistence of the disease, while no obvious changes in clinical course of the disease has been observed in negative patients. As a result, availability of this protein with characteristics of the naturally occurred antigen in high amounts and of the same purity is highly required for being utilized in medical research and diagnosis. At present, recombinant HBeAg is being produced in different expression systems for various applications. In the present study, by application of a simple expression/purification system in E. tali and with the aim of producing physiologically natural HBeAg, that portion of the gene, which exactly corresponds to amino acid sequence of the blood-borne HBeAg have been isolated and cloned into pQE-30 vector which adds a 6x His-tag to the N-terminal of the inserted gene, providing the ability of one step purification method by exploiting Ni-NTA column chromatography. Following restriction analysis and confirmation of recombinant vector accuracy, transformation to E. colt M15 cells and IPTG-mediated induction for protein expression was performed. Analysis of the purified protein by SDS-PAGE, ELISA and Western blotting indicated that obtained recombinant HBeAg had the perfect antigenic properties of authentic HBeAg and addition of the His-tag segment which produces extra epitopes on this antigen had no adverse effect on these properties. The results of this study clearly describe a very simple and efficient system for providing recombinant HBeAg in high yield and purity for diagnostic and physiological research applications.