Expression And Purification And Functional Analysis Of The Anti-hepatitis B Virus Polypeptide C20-MTS In Vitro

Background and Objective:Hepatitis B is a global infectious disease, which brings a serious threat to human and public health. Though a great progress has been made in hepatitis B prevention, there is still a lack of the excellently curative, low toxic and cheap drug for Hepatitis B treatment. So, It’s of great clinical significance to search new antiviral targets and develop new drugs with novel mechanisms. In our previous studies, a polypeptide sequence(C20/C20-MTS) targeting the core protein of hepatitis B virus was screened using the yeast two-hybrid system. In view of the fact that the C20/C20-MTS were produced mainly in the form of inclusion bodies, and it was difficult for us to obtain the active polypeptide using denaturation and renaturation method. In the present study, we try to express the C20/C20-MTS in the form of soluble form and test the virus inhibition of the polypeptide using Hep G2.2.15 cells, which will provide the basic data for development of the novel drug for Hepatitis B.Methods:First, we construct the prokaryotic expression vector of p MAL-c5x-C20/C20-MTS, transform it into the E. coli Rosatte(DE3) and evaluate the impact of the temperature, induction time and IPTG concentration on the expression levels of the target proteins. After purifying the recombinant protein by affinity chromatography, we evaluate the inhibition effect of the purified proteins on HBs Ag and HBc Ag in Hep G2.2.15 cells by the ELISA and Western blot technologies.Results:The p MAL-c5x-C20/C20-MTS recombinant plasmid was successfully constructed and the targeted proteins were mainly expressed in the form of solution. After the purification, we harvested the C20-MTS protein at 0.085 mg/ml and C20 at 0.093 mg/ml, and their purity were beyond 90%. The preliminary pharmacodynamics results indicated that the C20-MTS beyond 0.1 μg/ml could inhibit the virus replication and secretion and reduce the core protein level, and the highest inhibition was obtained at the level of 10 μg/ml.Conclusion:Our obtained C20-MTS protein in E. coli Rosatte cell can inhibit the replication of hepatitis B virus and reduce the core protein level in Hep G2.2.15 cells, which is expected to be a novel type polypeptide drug for anti-hepatitis B treatment.