Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli.
OBJECTIVE
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 SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein 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 Mycoplasma 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 p37 protein could directly bind to gastric tumor cell AGS.


CONCLUSIONS
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.
Ning
Jin-Ying
JY
Department of Biochemistry and Molecular Biology, School of Oncology and Beijing Institute for Cancer Research, Peking University, Beijing 100034, China.
Sun
Guo-Xun
GX
Huang
Su
S
Ma
Hong
H
An
Ping
P
Meng
Lin
L
Song
Shu-Mei
SM
Wu
Jian
J
Shou
Cheng-Chao
CC
D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D000918:Antibody Specificity; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001422:Bacterial Adhesion; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D015964:Gene Expression Regulation, Bacterial / Q000276:immunology; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009175:Mycoplasma Infections / Q000503:physiopathology; D045790:Mycoplasma hyorhinis / Q000472:pathogenicity; D010051:Ovarian Neoplasms; D013274:Stomach Neoplasms; D014774:Virulence
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial