Cloning And Expression Of MAP30 From Momordica Charantia And Its Antiviral Activity

Objective To prepare recombinant MAP30 from bitter melon,and investigate the intracellular transport of MAP30 in mammalian cell and the anti-HBV mechanism of MAP30.MethodsThe genomic DNA was exacted from seeds of mature bitter melon.After PCR amplification,the full-lenth gene of MAP30 was obtained.The DNA fragement was cloned into expression vector pET30a.The expression of the MAP30 in E.coli BL21 (DE3) was induced with 1 mM IPTG at 30℃for 3-5 h.The cells were harvested by centrifugation.After soncation,the cell lysate was centrifuged at 12000 rpm for 15 min. The column of Ni-NTA was used to affinitively purified the recombinant protein which contains his-tag.Two adult male rabbits(New Zealand) were simultaneously immunized with a mixure of 1 mg MAP30 and the same volume of complete freund's adjuvant in every 2 weeks.0.5 mg of the antigen mixed with an equal volume of incomplete freund's adjuvant was given subcutaneously a total of 4 times.Before immunization,blood sample were taken from the marginal vein of the rabbit ear and the sera were frozen as blank controls.The antiserum titer was measured by enzyme-linked immunoassay (ELISA) and the specificity was measured by Western bolt. Cell viability was evaluated by the MTT assay.Compare the toxicity of MAP30 on HBV-HepG2 cells and HepG2 cells using MTT.The low toxicity of MAP30,TCS and Lamivudine concentration was observed.At such concentration,the inhibition of MAP30 onto HBsAg and HBeAg was detected by ELISA,the levels of HBV DNA was detected by Real-Time PCR.We detected the DNase activity of MAP30 on pBluescript SK DNA in the presence of different ions.Hela cells were treated with 100μg/ml MAP30 and PBS(as control) for 45 min at 37℃.After endocytosis,the cells were harvested and encapsulated.Samples were prepared by TEM assay and low temperature assay.ResultsMAP30 gene was cloned by PCR,the sequence was confirmed same to that reported.After IPTG induction,SDS-PAGE showed a band with a molecular weight of 34kD.MAP30 were expressed in both soluble and inclusive body forms.Eluted with a series of different concentrations of imidazole in the column of Ni-NTA,the MAP30 was purified.The imidazole was removed using dialysis.The concentration of the recombinant protein was measured by Bradford method.The protein was saved in -20℃after filter sterilization.The antibody titer measured by ELISA method was about 128000.There were two bands showed in the map of Western bolt,with molecular weight of about 34kD and 67 kD(indicating the dimmer of the MAP30).The cytotoxicities of MAP30 in the tumor cells of Hela and HepG2 were much greater than that to the normal cells of LO2.The cytotoxicity of MAP30 in HepG2.2.15 is lower than that to HepG2 cells,the prototype.At a range of 0.1μg-100μg/ml concentration,the cytotoxicity of MAP30,TCS and Lamividine to HepG2.2.15 were below 15%.At those concentration,MAP30,TCS and Lamividine were able to inhibit the expression of HBsAg,HBeAg and HBV DNA duplication in HepG2.2.15 supernatant with dose-dependent.We found that MAP30 show different DNase activity on pBluescript SK DNA in the presence of different ions.MAP30 was located on the nucleus of the mamailian cells observed under immuno-electron microscopy.MAP30 can induced autophage.MAP30 may exert antiviral activity through enzyme activity on DNA in the nucleus of cells.ConclusionThe toxicity of MAP30 on the tumor cells was much greater than that to normal cells.After transfection with HBV,the cytotoxicity to HBV-HepG2 cells was lower than the toxicity of the prototype.At the low toxicity doses,MAP30 was able to inhibite the HBV DNA duplication.Targeting to the nucleus of cells might be important for MAP30 to function as antiviral agent.MAP30 may exert antiviral activity through enzyme activity on DNA in the nucleus of cells.MAP30 can induced autophage.