[ 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
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.
