RESUMO
AIM: To obtain monoclonal antibody against PIK3IP1 for further study of the structure and biological function of PIK3IP1 protein. METHODS: BALB/c mice were immunized with recombinant GST-PIK3IP1(62-168), Hybridoma cell lines secreting monoclonal antibodies against PIK3IP1 were screened by regular cell fusion and subcloning approach. The specificities of the monoclonal antibody was determined by ELISA, Western blot and Immunofluorescence assay. RESULTS: One hybridoma cell line (5C6) stable in secreting specific monoclonal antibody was successfully obtained. The subclass of IgG belonged to IgG1. The ascite titers of this monoclonal antibody reached 1:10(7). It could specifically bind to recombinant GST-PIK3IP1(62-168); protein and overexpressed PIK3IP1 and variant PIK3IP1-v1 proteins proved by Western blot. This antibody failed to react with E.coli lysates and glutathione S transferase (GST). At the same time, endogenous PIK3IP1 was not detected using 5C6 antibody. Immunofluorescence results revealed that overexpressed PIK3IP1 and variant PIK3IP1-v1 protein located in cytoplasm and distributed in fleck manner. CONCLUSION: Monoclonal antibody against PIK3IP1 with high titer and specificity has been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of PIK3IP1.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Membrana/imunologia , Animais , Escherichia coli/genética , Células HeLa , Humanos , Hibridomas , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , SolubilidadeRESUMO
OBJECTIVE: To obtain monoclonal antibodies against CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) for further study of the structure and biological function of CMTM7. METHODS: Three polypeptides were synthesized based on the bioinformatics analysis of the CMTM7 and coupled with keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with these mixed CMTM7 polypeptides. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Resulting hybridomas producing anti-CMTM7 antibodies were screened by enzymejlinked immunosorbent assay (ELISA). The specificities of these monoclonal antibodies were determined by Western blot, immunofluorescecence and immunocytochemistry (ICC). RESULTS: Two hybridoma cell lines (MC9 and 2C9) stable in secreting anti-CMTM7 monoclonal antibodies (MAbs) were generated. Both of them produced immunoglobulin G1 (IgG1) against CMTM7. 2C9 and MC9 recognized a region between amino acid residues 19-44 and 163-175 of CMTM7, respectively. MC9 antibody could be used for Western blot, immunofluorescecence and immunocytochmistry assay. However, 2C9 antibody could be only used for Western blot. Data obtained from immunofluorescence and ICC indicated that CMTM7 protein expression was upregulated in the early stage of lymphocyte activation treated with phytohemagglutinin (PHA). CONCLUSION: Monoclonal antibodies of high specificity against CMTM7 have been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of CMTM7.
Assuntos
Anticorpos Monoclonais/biossíntese , Quimiocinas/imunologia , Animais , Quimiocinas/genética , Humanos , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologiaRESUMO
OBJECTIVE: To obtain monoclonal antibodies against programmed cell death 10 (PDCD10) for further study of the structure and function of PDCD10 protein. METHODS: Balb/c mice were immunized with recombinant PDCD10, hybridoma cell lines secreting monoclonal antibodies against PDCD10 were screened by regular cell fusion and subcloning approach. The specificities of these monoclonal antibodies were determined by ELISA, Western blotting and Immunofluorescence assay. RESULTS: Three hybridoma cell lines (5G1, 4F7 and 3H5) stable in secreting specific monoclonal antibodies were successfully obtained. Subclass of IgG belonged to IgG1 (4F7 and 5G1) and IgG2b (3H5), respectively. The ascite titers of these monoclonal antibodies reached 1:10(7). They could specifically bind to recombinant PDCD10 and endogenous and overexpressed PDCD10 proteins proved by ELISA and Western blotting. They failed to react with E.coli lysates and glutathione S-transferase (GST). In addition, these three monoclonal antibodies could recognize different epitopes of PDCD10 proteins assessed by immune fluorescence competitive binding assay. Both endogenous and overexpressed PDCD10 protein mainly located in the nucleus. CONCLUSION: Monoclonal antibodies against PDCD10 with high titers and specificity have been successfully prepared, which has laid the foundation for further study of PDCD10 protein.
