[Cloning and expression of a novel gene restin and preparation of antisera against the recombinant restin].
OBJECTIVE
To clone a new human gene, restin, from retinoic acid-treated promyelocytic cell line HL-60, express the protein in E.coli and prepare the antisera against the protein.


METHODS
The restin gene was amplified from retinoic acid-treated promyelocytic cell line HL-60 by RT-PCR and cloned into a prokaryotic expression vector. The recombinant restin was induced to express in E. coli by temperature. After preliminary purification by SDS-PAGE, the restin protein was used to immunize rabbits to obtain antisera. The titers and specificity of the rabbit anti-restin antisera were tested by Western blot and immunofluorescence analysis.


RESULTS
Recombinant restin with M(r) being about 26,000 was highly expressed in E. coli. The titers of the anti-sera to restin ranged from 1:100 to 1:800. Immunofluorescence analysis showed that restin distributed mainly in the nuclei of COS-7 cells.


CONCLUSIONS
We successfully prepared the antisera against restin, which are useful for further investigation of biological functions of restin.
Lu
Fan
F
School of Life Science & Technology, Xi'an Jiaotong University, Xi'an 710048, China. lufan@fmmu.edu.cn
Yang
Hui
H
Wang
Xiao-gong
XG
Pu
Qin
Q
Li
Yi
Y
Zhao
Wen-ming
WM
Zhao
Zhong-liang
ZL
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D006801:Humans; D007106:Immune Sera / Q000032:analysis / Q000276:immunology*; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007106:Immune Sera; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C076744:cytoplasmic linker protein 170; D004262:DNA Restriction Enzymes