Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and A...Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.展开更多
AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light...AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light and heavy chains of the HBV preS2Ab gene,Ad315-preS2Ab,was constructed.Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expres-sion levels in vitro.Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells.ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells.The inhibitory effect of preS2Ab against hepatic carcinogen-esis was studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice.RESULTS:The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells,with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell.The expressed preS2Abs could recog-nize liver cells from HBV transgenic mice.ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV pa-tients than parental L02 cells expressing no preS2Ab.HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vec-tor or untreated mice.Additionally,the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase.CONCLUSION:Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells,and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.展开更多
The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The op...The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The open reading frame(ORF) encoding the CT440 protein from the C.trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase(GST) fusion protein in E.coli XL1-Blue.The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody.After verification by Western blot and immunofluorescence assay(IFA),the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells.Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection.The results showed that the hypothetical protein CT440 was localized in the C.trachomatis inclusion membrane,and was detectable 12 h after chlamydial infection.Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection.In summary,we have identified a new inclusion membrane protein that may be an important candidate for understanding C.trachomatis pathogenesis.展开更多
文摘Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.
基金Supported by The National Natural Science Foundation ofChina,No.30872998
文摘AIM:To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV in-fection and HBV-associated hepatic carcinogenesis.METHODS:An adenoviral vector carrying the full-length light and heavy chains of the HBV preS2Ab gene,Ad315-preS2Ab,was constructed.Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expres-sion levels in vitro.Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells.ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells.The inhibitory effect of preS2Ab against hepatic carcinogen-esis was studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice.RESULTS:The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells,with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell.The expressed preS2Abs could recog-nize liver cells from HBV transgenic mice.ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV pa-tients than parental L02 cells expressing no preS2Ab.HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vec-tor or untreated mice.Additionally,the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase.CONCLUSION:Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells,and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.
基金supported by the National Natural Science Foundation of China (Grant Nos. 30970165 and 81102230)the Hunan Provincial Natu-ral Science Foundation of China (Grant No. 09JJ3059)the Team Project for the Technology Innovation of Higher Education of Hunan Province,China
文摘The inclusion membrane proteins play potentially important roles in chlamydial biology and pathogenesis.Here we localized and characterized the hypothetical protein CT440 in Chlamydia trachomatis-infected cells.The open reading frame(ORF) encoding the CT440 protein from the C.trachomatis serovar D genome was cloned into the prokaryotic expression vector pGEX-6p and expressed as a glutathione-S-transferase(GST) fusion protein in E.coli XL1-Blue.The CT440 fusion protein was used to immunize mice to raise antigen-specific antibody.After verification by Western blot and immunofluorescence assay(IFA),the specific antibody was used to localize the endogenous CT440 protein and to detect its expression pattern in Chlamydia-infected cells.Cytosolic expression of CT440 in HeLa cells was also carried out to evaluate the effect of the CT440 protein on the subsequent chlamydial infection.The results showed that the hypothetical protein CT440 was localized in the C.trachomatis inclusion membrane,and was detectable 12 h after chlamydial infection.Expression of CT440 in the cytoplasm did not inhibit the subsequent chlamydial infection.In summary,we have identified a new inclusion membrane protein that may be an important candidate for understanding C.trachomatis pathogenesis.
