[Fusion expression of gastric cancer-related gene MPS-1 and preparation of rabbit antibody against GST-MPS-1].
OBJECTIVE
To express the fusion protein GST-MPS-1 in E.coli and prepare rabbit antibody against GST-MPS-1.


METHODS
MPS-1 cDNA was inserted into the vector of pGEX-5X. The recombinant was then transformed into E.coli BL21. After induction with the IPTG, the fusion protein GST-MPS-1 was expressed in E.coli. The purified fusion protein was then used to immunize the New Zealand rabbits to obtain the polyclonal antibody against GST-MPS-1. Specificity of the antibody was tested by Western blot and immunofluorescent staining.


RESULTS
The cDNA sequence of MPS-1 in the recombinant was confirmed by restriction endonuclease digestion and sequence analysis. SDS-PAGE result showed that fusion protein was highly expressed in E.coli with a molecular weight of 36 kDa. The titer of the rabbit serum against GST-MPS-1 was as high as 1x10(5) in ELISA analysis. The polyclonal antibody were found to specifically bind to MPS-1 protein in Western blot and immunofluorescent staining.


CONCLUSIONS
The preparation of the polyclonal antibody against MPS-1 lay a foundation for the further study of MPS-1 expression at protein levels and its function.
Wang
Yun-wei
YW
Department of Surgery, Ruijin Hospital, Shanghai Second Medical University, Shanghai Institute of Digestive Surgery, Shanghai 200025, China. w_yw1961@hotmail.com
Zhu
Zheng-gang
ZG
Gu
Qin-long
QL
Li
Jian-fang
JF
Qu
Ying
Y
Liu
Bing-ya
BY
Lin
Yan-zhen
YZ
D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics*; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D008856:Microscopy, Fluorescence; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; C075840:TTK protein, human