| Objectives1. To study the temporary expression of pICOSIg fusion protein expression vector in mammalian cells (COS7) and to identify the antigen specificity of its expressive product in vitro for the further study of its function.2. To study the stable expression of pICOSIg fusion protein expression vector in Chinese hamster ovary cell (CHO) cells and to purify its expression product and to identify the function of the fusion protein in vitro.3. To establish the model of the acute rejection in small bowel transplantation of rats and to study the expression of ICOS on the small bowel allografts during the period of acute rejection.Methods1. To identify the pICOSIg fusion protein expression vector by the double digestion of Kpn I and BamH I restrictive enzymes, and nucleic acid sequencingassay.Three days after pICOSIg were transfected into COS7 cells by DEAE-Dextran,the product was purified with protein A affinity chromatography and studied with SDS-PAGE and Western blotting to determine its molecular weight, purity, and antigen specificity.2. Expression vector pICOSIg and resistance plasmid pEGFP was cotransfected into CHO cells by electroporation.Then the selective medium containing G418 was employed to select positive clones. After repeating cloning culture and double antibody sandwich ELISA detection, the cell clones expressing ICOSIg stablely were obtained. After purified with protein A affinity chromatography,the pure fusion protein have been obtained,with which we study the function of the fusion proein by FCM and MLR in vitro.3. Heterotopic small bowel transplantation was performed with inbred SD and Wistar/A rats.48 recipients were divided into four groups equally: group I , Wistar/A( open-close operation); group II,Wistar/A→Wistar/A; group III,SD→ Wistar/A; groupIV, SD→Wistar/A+FK506.In each group two rats were killed on POD 3,5 and 7,and the donor intestine was resected under sterile condition.The samples were examined histologically by HE stains and the expression of ICOS was detected by immunohistochemistry technique.Results1. A 600 bp nucleic acid sequence was abtained after the pICOSIg fusion protein expression vector being digested by Kpn I and BamH I restrictive enzymes.Nucleic acid sequencing assay verified that the amplifed cDNA fragment was identical to mouse ICOS extracellular region.ICOSIg fusion protein expression vector was constructed and transfected into COS7 cells successfully. Supernatant was detected by double antibody (anti-ICOS-mAb,HRP-anti-hIg-mAb ) sandwich ELISA ,and the result was strongly positive.A protein band with molecular weightof 70kDa was found in the SDS-PAGE gel,which specifically bound with anti-human Fc monoclonal antibody labelled HRP in Western blotting analysis.2. We have got the CHO cell clones expressing ICOSIg fusion protein stablely and the fusion protein was available from the supernatant of cell culture medium after purified with protein A affinity chromatography. The protein could specifically bind with mouse spleen cells and could suppress the proliferation of T cells effectively in MLR,which depended on the concentration of the ICOSIg and the PODs.The suppression started on POD 1 and reached the peak on POD 3.3. We established the model of the acute rejection in small bowel transplantation of rats successfully.The histological examinination showed that mild acute rejetion occurred on POD 3,moderate acute rejection on POD 5,severe acute rejection on POD 7 in groupIII,while in group II and IV, only mild acute rejections were found on POD 7.The histologic evidence of group IV indicated that FK506 could effectively control acute rejection.The immunohistochemistry examinination showed that ICOS expression highly increased in group III on POD 3 and reach the peak on POD 5,significantly higher than that of other groups(P<0.05).ConclusionsFusion protein expression vector is successfully constructed,which containing extracellular region of mouse ICOS and human IgG Fc which was restricted by Kpn I and BamH I. Recombined mouse ICOSIg fusion protein was successfully and stablely expressed in CHO cells. Purified ICOSIg fusion with antigen specificity and immunocompetence are available which lays the foundation for the study of the mechanisms of ICOS/B7h costimulatory signals in transplantation.We establish the model of the acute rejection in small bowel transplantation of rats successfully.The immunohistochemistry examinination showed that ICOS...
|
PDF Full Text Request