Cloning And Expression Of A Novel Glycogene Glt25D2: The Relationship Between Glt25D2 And Hepatitis B Virus Large Envelope Protein

Objective:1.To clone and express of a Novel Glycogene Glt25D2 in Escherichiacol.and prepare the Glt25D2 specific rabbit polyclonal antibody.2.To observe the expression pattern of Glt25D2 in hepatocyte.3.To confirm the relation between Glt25D2 and LHBs.4.To demonstrate the effects of Glt25D2 expression downregulation with RNA interference,on the expression of LHBs.5.To indentificate the donor-substrate specificity of the Glt25D2 fused protein by Biacore analysis.Methods:1.Glt25D2,amplified from HepG2 cDNA,was ligated with pGEM-TEASY cloning vector. After verified the inserted fragment,Glt25D2 was cloned into pET-32a(+), inducible proeukaryotic expressive vector,and transformed into E.coli BL21 (DE3).The protein expression was induced with IPTG and analyzed with SDS-PAGE and western blot hybridization.The expressed product was purified and renatured by Ni affinity column chromatography.2.The purified GLT25D2 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody.The specificity and potency of polyclonal antibody were evaluated by western blot and ELISA.3.We constructed eukaryotic expression vector pEGFP-C1-GLT25D2,pDsRED-N1 -ERgic-53 and pDsRED-N1-LHBs to observe the expression pattern of Glt25D2, ERGIC-53,and LHBs with confocal laser scanning microscope.4.We confirmed the interaction between Glt25D2 and LHBs with co-immunoprecipitation and mammalian two-hybrid experiment.5.The Biacore analysis was used to determine the donor-substrate specificity of the Glt25D2.6.The RNA interference technique was used to explore the effect of Glt25D2 expression downregultion on LHBs expression. Results:1.Glt25D2 CDs was amplified from HepG2 cDNA.2.The Glt25D2 fusion protein was highly expressed.The protein production mainly was in inclusion body through SDS-PAGE analysis.3.The polyclonal antibody were obtained successfully.ELISA showed the titer of polyclonal antibody＞1:2560 000.The high specificity of the polyclonal antibody was testified with SDS-PAGE and western blot.4.Eukaryotic expression vector pEGFP-C1-GLT25D2,pDsRED-N1-ERGIC-53 and pDsRED-N1-LHBs were constructed and cotransfected into HepG2 cell line for 24-48h.We observed the expression pattern by confocal laser scanning microscope.The results showed that colocation relationship among Glt25D2, LHBs,and ERGIC-53.5.We constructed eukaryotic expression vector pcDNA3.1-Glt25D2,which was cotransfected into HepG2 cell line with the expressive vector of pcDNA3.1-LHBs. We confirmed the intra-cellular interaction between GLT25D2 and LHBS with co-immunoprecipitation.6.We constructed eukaryotic expression vector pACT-GLT25D2,which was cotransfected into HepG2 cell line with the expressive vector of pBIND-LHBs. We confirmed the intra-cellular interaction between Glt25D2 and LHBs with mammalian two-hybrid experiment.7.The Biacore analysis showed that Glt25D2 might bind with NeuAc and Ca²⁺.8.The shRNA vector were constructed sucessfully.The most effective shRNA expression vector transfected into HepG2.2.15 cell line.The results demonstrated downregulation of Glt25D2 expression might inhibite the expression of LHBs.Conclusion:1.The GLT25D2 fusion protein(90 kDa) was expressed successfully.2.The specific polyclonal antibody of Glt25D2 had high bioactivity.3.The methods of Co-immunopercipitation,mammalian two-hybrid and confocal observation confirmed that the interaction between GLT25D2 and LHBs,which put forward some new clues for studying the molecular biology mechanism of Glt25D2 with LHBs.4.Glt25D2 might bind with NeuAc and Ca2+,and might play a role in LHBs O-glycosyaltion.5.Downregulation of Glt25D2 expression might inhibite the expression of LHBs.