[Expression of midkine fusion protein and preparation and application of its monoclonal antibodies].
OBJECTIVE
To express midkine (MK) protein, prepare its monoclonal antibody (mAb), and detect its expression in tumor cells.


METHODS
The human MK gene fragment was cloned from kidney cancer tissues by RT-PCR and inserted into the prokaryotic expression vector pMS-31b. The MS2-MK fusion protein was expressed in E.coli after temperature induction. The purified fusion protein was used to immunize BALB/c mice to prepare mAbs against MK by routine hybridoma technique. The Ig subclasses of the mAbs were identified by ELISA. The expression of MK in tumor cells was detected with the mAbs.


RESULTS
MK gene was cloned and MS2-MK fusion protein was expressed successfully. Two hybridoma cell lines secreting anti-MK mAbs were obtained. The Ig subclass of the 2 mAbs was IgG1 and IgG2a, respectively. MK was expressed in gastric carcinoma cell line MGC803 and gastric cancer tissue.


CONCLUSIONS
The MS2-MK fusion protein was expressed in E.coli and the mAbs against MK were pepared, which allow futher research on MK function.
Zhang
Qing-yun
QY
Department of Immunology, Beijing Institute for Cancer Control, The School of Oncology, Peking University, Beijing 100036, China. qingyzhang@btamail.net.cn
Yang
Min
M
Wang
Ya-ming
YM
Xu
Jian-jun
JJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000378:metabolism / Q000473:pathology; D007074:Immunoglobulin G / Q000276:immunology; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D000078792:Midkine; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000911:Antibodies, Monoclonal; D016207:Cytokines; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D000078792:Midkine