Expression and purification of spindlin 1,a novel cancer related protein,and preparation of its polyclonal antibody
10.3867/j.issn.1000-3002.2014.03.002
- VernacularTitle:肿瘤相关蛋白spindlin 1的表达、纯化及多克隆抗体的制备
- Author:
Lin CHEN
;
Quan ZENG
;
Peng ZHANG
;
Jingxue WANG
;
Lipeng QIN
;
Yang LYU
;
Xue NAN
;
Wen YUE
;
Xuetao PEI
- Publication Type:Journal Article
- Keywords:
spindlin 1;
protein biosynthesis;
polyclonal antibody;
ovarian neoplas ms
- From:
Chinese Journal of Pharmacology and Toxicology
2014;(3):321-328
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE Toprepareapolyclonalantibodyforspindlin1protein,anovelcancer related protein,and to provide the data for a better understanding of its functions and screening tu mor. METHODS Purifiedspindlin1proteinwasinjectedintorabbitstoproducethepolyclonalantiserumafter removing glutathione S-transferase (GST)from the fusion protein spindlin 1-GST that was expressed in Escherichia coli..The antiserum was purified through the Hitrap Protein A system,and the titer of spin-dlin 1 polyclonal antibody was detected by ELISA.The specificity of the polyclonal antibody was deter-minedbyWesternblottingandimmunohistochemistry.RESULTS Thetiterofspindlin1polyclonalanti-body was 1∶2000.Western blotting detection demonstrated that the spindlin 1 polyclonal antibody recog-nized myc-spindlin 1 reco mbinant fusion protein in HeLa cells transfected with pAdeasy-myc-spindlin 1 , which also corresponded with Myc.antibody.The HeLa cells were transfected with enhanced green fluo-rescence protein (EGFP)and spindlin 1 vector(pEGFP-C3-spindlin 1 ),which was confirmed by the in-dependent GFP fluorescence assay.The results of immunohistochemistry detection with the spindlin 1 polyclonal antibody suggested that spindlin 1 was mainly expressed in the nuclei of HeLa cells.More i m-portantly,in i mmunohistoche mical assays,the spindlin 1 antibody recognized nuclear spindlin 1 expres-sioninclinicalovariancancertissues.CONCLUSION Thespecificspindlin1polyclonalantibodyispre-pared,which may be used to detect cancer-related protein spindlin 1 in HeLa cells and ovarian cancer tissues.
