To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30...To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.展开更多
Background:Human epidermal growth factor receptor(EGFR)is an oncogenic gene and one of top targets of precision therapy in lung cancer with EGFR mutations.Although there are many reports for some individual cancers,co...Background:Human epidermal growth factor receptor(EGFR)is an oncogenic gene and one of top targets of precision therapy in lung cancer with EGFR mutations.Although there are many reports for some individual cancers,comprehensive profiling of EGFR mutations,overexpression,amplification,DNA methylation,and their clinical associations across many different cancers simultaneously was not available.This study aimed to fill the gap and provide insights to the alteration spectrum of EGFR and its therapeutic and prognostic implications.Methods:The Cancer Genome Atlas(TCGA)datasets for 32 cancer types involving 11,314 patients were analyzed for alterations(mutations and amplification/deletion),abnormal expression and DNA methylation in EGFR gene.Mutation frequency,genomic location distribution,functional impact,and clinical targeted therapy implication were compared among different cancer types,and their associations with patient survival were analyzed.Results:EGFR alteration frequency,mutation sites across functional domains,amplification,overexpression,and DNA methylation patterns differed greatly among different cancer types.The overall mutation frequency in all cancers combined was relatively low.Targetable mutations,mainly in lung cancer,were primarily found in the Pkinase_Tyr domain.Glioblastoma multiforme had the highest rate of alterations,but it was dominated by gene amplification and most mutations were in the Furin-like domain where targeted therapy was less effective.Low-grade glioma often had gene amplification and increased EGFR expression which was associated with poor outcome.Colon and pancreatic adenocarcinoma had very few EGFR mutations;however,high EGFR expression was significantly associated with short patient survival.Squamous cell carcinoma regardless of their sites(the head and neck,lung,or esophagus)exhibited similar characteristics with an alteration frequency of about 5.0%,was dominated by gene amplification,and had increased EGFR expression generally associated with short patient survival.DNA me展开更多
基金supported by Science and Technology Commission of Shanghai Municipality(03JC14085)grant of National Natural Science Foundation#30170888the grant from E-institutes of Shanghai Universities Immunology Division.
文摘To prepare monoclonal antibody specific to epidermal growth factor receptor(EGFR)intracellular domain,its gene was amplified from total RNA of A431 cell by RT-PCR.Then the gene was cloned into prokaryotic vector pET30a(+).The recombinant plasmid was transformed into E.coli BL21(DE3)strain for protein expression. Recombinant protein was induced with IPTG and purified using Ni^(2+)NTA agarose.Then the anti-EGFR monoclonal antibody(mAb)was prepared with classical hybridoma technique.Positive clones were selected using indirect enzyme-linked immunoabsorbent assay(ELISA).Totally 4 hybridoma clones were obtained and these mAbs were IgGl(3 clones)and IgG2a(I clone),respectively.Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line.The mAbs will be useful in the study of EGFR-mediated signal transduction.Cellular & Molecular Immunology.2004;1(2):137-141.
基金China Scholarship Council,Grant/Award Number:201806015028Chinese National Natural Science Foundation,Grant/Award Numbers:81101998,81872018,81372292+1 种基金Chinese Ministry of Science and Technology,Grant/Award Number:2017YFC0110200Mayo Clinic Center for Individualized Medicine。
文摘Background:Human epidermal growth factor receptor(EGFR)is an oncogenic gene and one of top targets of precision therapy in lung cancer with EGFR mutations.Although there are many reports for some individual cancers,comprehensive profiling of EGFR mutations,overexpression,amplification,DNA methylation,and their clinical associations across many different cancers simultaneously was not available.This study aimed to fill the gap and provide insights to the alteration spectrum of EGFR and its therapeutic and prognostic implications.Methods:The Cancer Genome Atlas(TCGA)datasets for 32 cancer types involving 11,314 patients were analyzed for alterations(mutations and amplification/deletion),abnormal expression and DNA methylation in EGFR gene.Mutation frequency,genomic location distribution,functional impact,and clinical targeted therapy implication were compared among different cancer types,and their associations with patient survival were analyzed.Results:EGFR alteration frequency,mutation sites across functional domains,amplification,overexpression,and DNA methylation patterns differed greatly among different cancer types.The overall mutation frequency in all cancers combined was relatively low.Targetable mutations,mainly in lung cancer,were primarily found in the Pkinase_Tyr domain.Glioblastoma multiforme had the highest rate of alterations,but it was dominated by gene amplification and most mutations were in the Furin-like domain where targeted therapy was less effective.Low-grade glioma often had gene amplification and increased EGFR expression which was associated with poor outcome.Colon and pancreatic adenocarcinoma had very few EGFR mutations;however,high EGFR expression was significantly associated with short patient survival.Squamous cell carcinoma regardless of their sites(the head and neck,lung,or esophagus)exhibited similar characteristics with an alteration frequency of about 5.0%,was dominated by gene amplification,and had increased EGFR expression generally associated with short patient survival.DNA me
