Cloning Of WCD4 And Antibody Production

Objective1. Cloning woodchuck and Chinese woodchuck CD4 molecules. Preparation of polyclonal and monoclonal antibodies.2. To establish a mouse and marmot lymphocyte proliferation test detection methods.Methods1. Cloned the cDNA sequence of CD4 from woodchuck PBMC, and then sequenced and compared with other mammals CD4 amino acid sequence and nucleotides sequence.2. Cloned the cDNA sequence of CD4 from China's woodchuck PBMC, sequenced and compared with the analysis of wCD4 and cwCD4 sequences.3. From Marmot liver tissue or in freshly isolated PBMC, using proteinase K digestion and phenol/chloroform extraction method to obtain DNA. PCR amplification of Marmot cytochrome b gene and analysis phylogenetic.4. Real-time fluorescence quantitative PCR detection of intrahepatic CD4 expression level in WHV infected marmot.5. Polyclonal antibodies anti-CD4 were developed by immunizing rabbits with purified recombinant protein, and the sensitivity and specificity were tested by ELISA, immunofluorescence and Western blot assays. 6. Balb/c mice were immuned following a routine procedure by GST-CD4(390) or GST-CD4(189), adjuvated with Al(OH)3. Positive clones of conjugated cells from mice myeloma cell SP2/0 and immuned spleen cell were screened out through ELISA and IF. Supernate of cultured hybridoma cells was collceted, and Ig subtypes of the mAb were determined as a consequence. Ascites were induced in mice after injection of the hybridoma cells, and antibody titer within both the ascties and supernate were determined by ELISA. Mice ascites were purified by Ammonium sulfate precipitated.7. The sensitivity and specificity of monoclonal antibody were tested by ELISA, immunofluorescence and Western blot assays.8. Using monoclonal anti-wCD4 to detection of Marmota PBMC and intrahepatic CD4+ T lymphocytes by Western blot, IF IHC.9. Using MTT assay method and CFSE to assess cell proliferationResults1. We have got the sequences of woodchuck CD4 and Chinese woodchuck CD4. Homology of wCD4 sequences with the respective sequences of other species on the nucleotide level homology was People75.33%, chimpanzee 76.00%, rabbits73.13%, cats72.39%, pigs71.29%, dogs71.15%, mice69.09%, rats69.31%, ducks53.89%, chickens52.79% respectively.2. The CD4 nucleotide homology of woodchuck and Chinese woodchuck is 99.6%.3. Through phylogenetic analysis, we found a high degree homology of woodchuck and Chinese woodchuck.4.4. RT-PCR assay, intrahepatic CD4 mRNA express to the peak in the second week after WHV infection.5. The polyclonal antibody against wCD4 was developed successfully, and the antibody had both high sensitivity and high specificity. 6. Four cell strains named as Gl,G2,C9,C10, had the ability of stable secretion of specified mAb, the subtypes of which were classified into IgGl. Stable capability of cell proliferation and antibody secretion could be maintained even if cultured for a consecutive six months, adapted to less regular serum and repeatedly frozen and resuscitated. Antibody titers of supernant fluid and ascites count for 103～104 and 107～108 respectively.7. Using Western blot, IF approach, the four monoclonal antibodies are able to recognize wCD4 molecules which was expression in the transfected wCD4 BHK. Because of the four antibodies can be used for WB, IF detection, it concluded that the four antibodies recognize CD4 molecules should be the natural conformation of the linear epitopes.8. Using IF, IHC method, the four antibodies are able to identify the Marmot PBMC CD4 +T cells.9. Using IHC methods, found that after marmots WHV infection, intrahepatic CD4+ T-cell infiltration increased significantly at the fifth week.10. Successfully establishment MTT assay method and CFSE to assess cell proliferation.Conclusions1. Successfully cloned wCD4, and cwCD4, then found they had 99.6% homology.2. Successfully prepared anti-woodchuck CD4 polyclonal antibody which had both high sensitivity and high specificity.3. Successfully prepared anti-CD4 monoclonal antibody, which could identify the Marmot PBMC of CD4+ T cells.