Construction Of Prokaryotic Expressionvector Of CTLA4 In Woodchuck And Polyclonal Antibody Production

BackgroundWoodchuck hepatitis virus(WHV) is closely to HBV, having higher similarities in morphology, genome structure and gene products ,replication, epidemiology, and the course of infection. So the woodchuck is an very important animal model to investigate the pathogenesis of hepatitis B virus (HBV) infection in humans. Most research aimed at understanding the mechanisms underlying recovery from HBV infection has been focused on the factors that lead to the activation of antiviral T-cell responses. In fact, the outcome of HBV infection varies according to the vigor of the immune response, a process that is also regulated by a number of molecules, including the cell surface receptor CTLA-4, which is an inhibitory receptor expressed by T lymphocytes that acts largely as a negative regulator of T-cell responses. Objective1. to construct a prokaryotic plasmid expressing of CTLA4 in woodchuck2.purify the recombinant protein, and to develop polyclonal antibodiesMethods1. DNA fragment coding CTLA4 was cloned into PGTK-2XF vector using gene engineering technique2. Recombinant plasmid was transformed into host strain BL21 and then protein expression was induced3. Recombinant protein was purified by electroeluting4. Polyclonal antibodies were developed by immunizing rabbits with purified recombinant protein ,,and the sensitivity and specificity were tested by EL ISA , immunofluorescence and Western blot assaysResults1. CTLA4 was cloned into vector PGTK-2XF ,then expressed in E. coli Induced by IPTG,the recombinant protein was purified using electroeluting2. polyclonal antibody was developed by immunizing rabbit with the recombinant protein...