[Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody].
OBJECTIVE
To clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer.


METHODS
Based on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-1 with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL(21) cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines.


CONCLUSIONS
The migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.
Gong
Wei
W
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. gwei203@ yahoo.com.cn
Li
Jie
J
Wang
Yun-Ling
YL
Nan
Qing-Zhen
QZ
Jiang
Bo
B
Zhang
Hong-Quan
HQ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000276:immunology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000276:immunology*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C473794:FBLIM1 protein, human; D011993:Recombinant Fusion Proteins