Generation Of Monoclonal Antibodies Specifically Against The Antigen Of DIXDC1 And Study Of The Distribution Of DIXDC1 Protein In Human Colon Carcinoma Tissues

Purpose To prepare specific anti-DIXDC1 monoclonal antibody,then exam ine the characteristics of the generated antibody,and identify the expression of DIXDC1 protein in colon tumors and related normal tissues,and hope to provide preliminarily theoretical basis for the further study of relations between DIXDC1 and genesis of colon tumors.Methods The recombinant plasmids,pGEX-DIXDC1-C and pGEX-DIXDC1-N which encoding the C-terminus or N-terminus region of DIXDC1 protein, were transformed separately into E.coli BL21(DE3).After being expressed by inducing with IPTG,both of the fusion proteins(GST-DIXDC1-N and GST-DIXDC1-C) were purified by GST sepharose affinity chromatography,and then were used to immunize female Balb/c mice.Spleen cells from immunized Balb/c mice with the highest titer were fused with mouse myeloma cells(SP2/0) according to the standard hybridoma technique.The hybridomas were primarily screened by indirect enzyme-linked immunosorbent assay(ELISA),and the positive clones were further verified by Western blot.Finally,to study the expression and localization of protein DIXDC1 in colon tumors tissues and related normal tissues, immunohistochemistry(SP) was performed by using the prepared anti-DIXDC1 monoclonal antibody.Results We successfully expressed and purified recombined GST-DIXDC1 protein.After being screened by ELISA and Western Blot,ten hybridoma clones secreting anti-DIXDC1 monoclonal antibody with a high degree were obtained. Besides that,eight of them could be used in immunohistochemistry.DIXDC1 protein was not only overexpressed in the malignant endothelial cells of colorectal carcinoma compared with normal sections of themselves,but also in smooth muscle cells of colorectum.Conclusions Monoclonal antibody specifically against protein DIXDC1 was successfully developed.Eight hybridoma clones could recognize DIXDC1 not only in the denatured form but also in the native form.By using the MAbs,we found DIXDC1 protein was overexpressed in human colon carcinoma tissues.