[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombina...[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombinant plasmids containing the amplified fragment were constructed. After prokaryotic expression and purification, the obtained recombinant protein was used to immunize Balb/c mice. Finally, the spleen cells were fused with myeloma cells （SP2/0）, and antibody titer of culture supematant was detected by ELISA. [ Result] A 510-bp gene fragment was amplified by PCR. The recombinant plasmid pET-32a-CD8 alpha was transformed into E. coli, and 39 kDa His-CD8 alpha fusion protein was induced to expression. After subcloning, the culture supernetant was detected by ELISA. A hybridoma cell strain, which could stably excrete antibody against CD8 alpha protein, was obtained and named Cll. The ELISA titer of cell supematant was higher than 1 ： 640. [ Conclusion] A hybridoma cell strain has been established using the CD8 alpha expressed in prokaryoUc system as immunogen.展开更多
[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha ...[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha chain of MHC II and exons 3 -6 encoding beta chain of MHC II were performed based on its protein sequences. After BALB/c mice were immunized with the purified fusion proteins, the mouse spleen cells were fused with mouse myeloma cells SP2/0. Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained. These three hybridoma cell strains were named as MHC II alpha-4, MHC II betas-2 and MCH II betas-31, respectively. Their titers of ascites in indirect ELISA were 1 ： 256 000, 1 ： 256 000 and 1 ： 1 280 000, respectively. These antibodies could specifically recog- nize MHC II alpha chain or beta chain in western blotting. [ Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.展开更多
[ Objective] To prepare monoclonal antibodies against chicken immunoglobulin G （IgG） and improve the diagnostic level of specific antibodies in chickens. [ Method] Chicken IgG was isolated by saturated ammonium sulf...[ Objective] To prepare monoclonal antibodies against chicken immunoglobulin G （IgG） and improve the diagnostic level of specific antibodies in chickens. [ Method] Chicken IgG was isolated by saturated ammonium sulfate and purified by Sephadex G-200 column chromatography. Then the BALB/c mice were immunized by the chicken IgG, and the spleen cells were fused with mouse myeloma cells SP2/0. Finally, the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] Four hybridoma cell strains secre- ting monoclonal antibodies against chicken IgG were obtained and named as C44, C45, C67 and C68, and their ascites titers in indirect ELISA were 1 ： 640 000, 1 ： 320 000, 1 ： 640 000 and 1 ： 80 000, respectively. The monoclonal antibodies secreted by C44 and C45 could recognize light chains of chicken IgG and those secreted by C,67 and C68 could recognize heavy chains of chicken IgG. They all could not recognize IgG from duck, rabbit and swine. Additionally, the Ig type identification results showed that they all belonged to IgGl. [ Conclusion] Four cell strains of obtained hybridoma can stably produce the monoclonal antibodies against chicken IgG.展开更多
基金supported by the grants of the National Natural Science Foundation of China (30671537)
文摘[ Objective] The aim of the study was to obtain monoclonal antibody against chicken CD8 molecule. [ Method] A fragment of chicken CD8 a/pha gene was amplified by PCR with a pair of designed primers. Then two recombinant plasmids containing the amplified fragment were constructed. After prokaryotic expression and purification, the obtained recombinant protein was used to immunize Balb/c mice. Finally, the spleen cells were fused with myeloma cells （SP2/0）, and antibody titer of culture supematant was detected by ELISA. [ Result] A 510-bp gene fragment was amplified by PCR. The recombinant plasmid pET-32a-CD8 alpha was transformed into E. coli, and 39 kDa His-CD8 alpha fusion protein was induced to expression. After subcloning, the culture supernetant was detected by ELISA. A hybridoma cell strain, which could stably excrete antibody against CD8 alpha protein, was obtained and named Cll. The ELISA titer of cell supematant was higher than 1 ： 640. [ Conclusion] A hybridoma cell strain has been established using the CD8 alpha expressed in prokaryoUc system as immunogen.
基金supported by the National Natural Science Foundation (30671537)
文摘[ Objective] To prepare the monoclonal antibody against chicken major histocompatibility complex class II molecules （MHC II）. [ Method ] The prokaryotic expression of the gene fragments of exons 2 -6 encoding alpha chain of MHC II and exons 3 -6 encoding beta chain of MHC II were performed based on its protein sequences. After BALB/c mice were immunized with the purified fusion proteins, the mouse spleen cells were fused with mouse myeloma cells SP2/0. Then the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] One hybridoma cell strain secreting monoclonal antibody against alpha chain and two strains secreting monoclonal antibody against beta chain were obtained. These three hybridoma cell strains were named as MHC II alpha-4, MHC II betas-2 and MCH II betas-31, respectively. Their titers of ascites in indirect ELISA were 1 ： 256 000, 1 ： 256 000 and 1 ： 1 280 000, respectively. These antibodies could specifically recog- nize MHC II alpha chain or beta chain in western blotting. [ Conclusion] Three obtained hybridoma stains can stably produce the monoclonal antibody against chicken MHC class II molecules.
基金supported by the National Natural Science Fund (30671537)
文摘[ Objective] To prepare monoclonal antibodies against chicken immunoglobulin G （IgG） and improve the diagnostic level of specific antibodies in chickens. [ Method] Chicken IgG was isolated by saturated ammonium sulfate and purified by Sephadex G-200 column chromatography. Then the BALB/c mice were immunized by the chicken IgG, and the spleen cells were fused with mouse myeloma cells SP2/0. Finally, the positive hybridoma cells were screened and detected by indirect enzyme-linked immunosorbent assay （ELISA）. [ Result] Four hybridoma cell strains secre- ting monoclonal antibodies against chicken IgG were obtained and named as C44, C45, C67 and C68, and their ascites titers in indirect ELISA were 1 ： 640 000, 1 ： 320 000, 1 ： 640 000 and 1 ： 80 000, respectively. The monoclonal antibodies secreted by C44 and C45 could recognize light chains of chicken IgG and those secreted by C,67 and C68 could recognize heavy chains of chicken IgG. They all could not recognize IgG from duck, rabbit and swine. Additionally, the Ig type identification results showed that they all belonged to IgGl. [ Conclusion] Four cell strains of obtained hybridoma can stably produce the monoclonal antibodies against chicken IgG.
