[Prokaryotic expression, polyclonal antibody preparation and subcellular localization of CIB].
OBJECTIVE
To construct the prokaryotic expression vector pET32a-CIB, prepare the specific polyclonal antibody against CIB and study the subcellular localization of CIB.


METHODS
CIB was amplified by RT-PCR from human brain tissue and cloned into prokaryotic expression vector pET32a-CIB. The CIB fusion protein was expressed in BL21 (DE3)/pET system and identified by SDS-PAGE. The mice were immunized with the polyacrylamide gel particles containing the CIB fusion protein for polyclonal antibody preparation. The antibody was purified by affinity chromatographic column matrix coupled with protein G, antigen respectively and then identified by Western blot and immunohistochemistry.


RESULTS
The protein of CIB was obtained by recombination expression. The specificity of polyclonal antibody was obtained by immunizing BALB/c mice with polyacrylamide gel particles containing the fusion protein of CIB and purification. The results of immunohistochemistry demonstrated that CIB was localized predominantly in the cytoplasm of SHG44 and Hhu7 cells.


CONCLUSIONS
The protein of CIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of CIB has been obtained, which can be used for further research into the function of CIB.
Shi
Jie
J
Department of Pathology and Anatomy, Lanzhou University, China.
Hui
Ling
L
Zhang
Xu
X
Wang
Xiao-Hui
XH
Yang
Jin-Sheng
JS
Lu
Tong-de
TD
Zhao
Zhi-Hua
ZH
Ha
Xiao-Qin
XQ
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000906:Antibodies; C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins