Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

查看全文 作　　者：Jin-Ying Ning Guo-Xun Sun Su Huang Hong Ma Ping An Lin Meng Shu-Mei Song Jian Wu Cheng-Chao Shou, Department of Biochemistry and Molecular Biology, School of Oncology and Beijing Institute for Cancer Research, Peking University, Beijing 100034, China

出　　处：《World Journal of Gastroenterology》索引2003年第9卷第10期,共5页

基　　金：Key Project of National Natural Science Foundation of China,No.30130190;Beijing Natural Science Foundation,No.7012007;Oncology Key Program and Cancer Center of Peking University 摘　　要：AIM: To clone and express the antigen of monoclonal antibody (Mab) PD4 for further investigation of its function.METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDSpolyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Nycoplasma hyorhinis (M. Hyorhinis) was further confirmed with Western blot analysis by infecting M. Hyorhinis-free HeLa cells and eliminating the M. Hyorhinis from MGC803cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations.Tmmunofluorescence assay was used to demonstrate if p37protein could directly bind to gastric tumor cell AGS.RESULTS: The cDNA library constructed with MGC803 cells was screened by Mab PD4 as probes. Unfortunately, the positive clones identified with Mab PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasrna hyorhinis (M. Hyorhinis)by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. Hyorhinis from MGC803 cells and by infecting M. Hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37protein could directly bind to gastric tumor cell AGS.CONCLUSION: The antigen recognized by Mab PD4 is from M. Hyorhinis, which suggests the actions involved in Mab PD4 is possibly mediated by p37 protein or M. Hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation. 关键词：大肠埃希氏杆菌单克隆抗体抗原 PCR 基因探针免疫沉淀反应 PD4

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Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

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