Generation And Identification Of Monoclonal Antibodies Against Chiken Tumor Suppressor P53 And Its Target Gene P21

Deoxyribonucleic acid(DNA)is the main hereditary substance of biological object,composed of deoxyribose sugars and four nitrogen-containing nucleobases.Sress signals,such as UV irradiation,X ray,virus infection and chemicals,can induce DNA damage,including substitutation,deletion,insertion and inversion,leading to DNA dysfunction and activation of various cellular responses.Accumulation of DNA damage can induce cell death,tissue damage and malfunction,immune disorder,impaired stem cell regeneration,cancers and other diseases.Therefore,exploring the DNA damage response pathway is of great interest.p53,an transcript factor,is involved in regulating important physiological and pathological processes,including cell growth,cell differentiation,celllular senescence,apoptosis,immune response and tumorigenesis.In normal cells,the expression level of p53 protein is exremely low.However,in response to exogenous and endogenous stimuli,such as hypoxia,DNA damage and oncogene activation,the expression level of p53 protein is dramatically elevated and acts as an immune checkpoint and genome guardian.p53 regulates a large number of target genes,including cyclin-dependent kinase inhibitor p21.Upon DNA damage,activated p53 induces the accumulation of p21,which binds to and inhibits the activity of cyclin-CDK complexes,and consequently leads to cell cycle arrest to block the replication of damaged DNA.Although much has been learned about human and mammalian p53,chicken p53 and its fuction in DNA damage response pathway is unknown.And there are no effective antibodies against chicken p53 and p21.In this study,chicken p53 and p21 genes were amplified by PCR,and expressed.In addition,using monoclonal antibody preparation method,monoclonal antibodies against chicken p53 and p21 were successfully generated.This study contains two research chapters:1.Generation of monoclonal antibodies against chicken p53RNA was extracted from CEF cells,and cDNA was obtained by RT-PCR.p53 gene was amplified from the cDNA,and subcloned into pET,30a vector.The recombinant plasmid named as pET-30a-p53,and transformed into E.coli BL21.Being induced by IPTG,the p53 protein was expressed and analyzed by SDS-PAGE.Results showed that p53 was expressed both in supernatant and inclusion body.Balb/c mice were immunized with the prepared p53 antigen.The antibody levels were detected by Western blot(WB),indirect immunofluorescence assay(IFA),enzyme-linked immunosorbent assay(ELISA).Results showed that chicken p53 could be specifically recognized by p53 anti-sera.Monoclonal antibodies against p53 were obtained by hybridoma technique,Six positive hybridoma clones were identified as 1D7,3C4,4D4,4F6,4F7 and 4H2,the subtype of which was IgG.1D7 was amplified to immunize mice,and the ascites fluid was collected.2.Generation of monoclonal antibodies against chicken p21The p21 gene was amplified by PCR,and subcloned into the prokaryotic expression vector,pET-30a.The recombinant plasmid,designated as pET-30a-p21,was transformed into E.coli BL21(DE3)competent cells.Being induced by IPTG,the p21 protein was expressed and analyzed by SDS-PAGE.The target bands were cut,ground and used as antigens to immunize Balb/c mice.The anti-sera were collected,and detected by Western blot(WB),indirect immunofluorescence assay(IFA),enzyme-linked immunosorbent assay(ELISA).The spleen was isolated and fused with SP2/0.Hybridoma clones were screened and twelve positive hybridoma clones were identified as 1B12,1E12,1H7,2B5,2C5,C8,2D8,2F2,3C11,3D6,G7,and 4G6,the subtype of which was IgG.