The Expression Separation And Purification Of Extracellular Domain Ⅰ～Ⅲ Of Intracellular Adhesion Molecule-1

Intercellular adhesion molecule-1(ICAM-1) is a kind of glucoprotein on cell surface and concerned with recognition of antigen,combination with complement and cell adhesion.ICAM-1 sheds from the cell surface to form the soluble intercellular adhesion molecule-1(sICAM-1).The abnormal expression of sICAM-1 is one of the important factors in Graves' disease(GD) and the level of serumal sICAM-1 has significant value in judging the relapse of GD and stopping drug administration.The immunologic function of ICAM-1 is main concentrated on extracellular domainⅠ～Ⅲof ICAM-1.Our lab has cloned the gene of extracellular domainⅠ～Ⅲof ICAM-1 and expressed the relevant fusion protein.Besides we have also produced the rabbit against human polyclonal antibody of ICAM-1.There is commodity ICAM-1 in forerign countries which is unstable,espensive and can not satisfy the purity of isotopic labeling.If we could acquired the pure protein of extracellular domainⅠ～Ⅲof ICAM-1,then we can use the antigen to establish a ultramicro analysis technique in detecting the serum of human sICAM-1.It will consistent with the current situation of our country and has economics significance and clinical application prospect.Objective:1.The fusion protein of extracellular domainⅠ～Ⅲof ICAM-1 in prokaryotic system by using the recombinant plasmid ZpET42aICAM preserved by our lab.After purification,the fusion protein was digested by thrombin,then we can obtain the protein of extracellular domainⅠ～Ⅲof ICAM-1 by immunoaffinity chromatography and high performance liquid Chromatography.2.At the same time the fusion protein of His Tag-ICAM-1 in prokaryotic system by using the recombinant plasmid ZpET28aICAM preserved by this research.,then we can obtain the protein of extracellular domainⅠ～Ⅲof ICAM-1.Methods:1.The recombinant plasmid ZpET42aICAM was transformed into E.Coli BL21(DE3).The bacteria were induced by IPTG.(isopropylβ-D-thioglactoside).The fusion protein purified by His.Tag purification system, we can get the purified recombinant GST-ICAM-1.Then we cut the GST.Tag from the fusion protein by the enzymolysis of thrombin,The purified product was identified by SDS-PAGE.Then we extracted the purified rabbit against human ICAM-1 IgG from the rabbit against human ICAM-1 serum by saturated ammonium sulfate and then the Protein A Sepharose CL-4B resin.After that we can get the purified protein of ICAM-1 by CNBr-activited Sepharose CL-4B gel immunoaffinity chromatography,at the same time HPLC was applied in the course of Separation and Purification of extracellular domainⅠ～Ⅲof ICAM-1.2.The recombinant plasmid of ZpET28aICAM was transformed into E.Coli BL21(DE3) which was renew constructed.The bacteria were induced by IPTG., After purification of the fusion protein by His.Tag purification system,we can get the purified recombinant His-ICAM-1.the purification result,the molecular weight, and the quantity of purified recombinant His-ICAM-1 were identified by SDS-PAGE,and Comasse Brilliant Blue method.The immunological activity of the fusion protein was identified by Western Blot.Results.1.The Expression Separation and Purification of Extracellular domainⅠ～Ⅲof GST-ICAM-1 by immunoaffinity chromatographyThe recombinant plasmid of ZpET42aICAM was transformed into E.Coli BL21(DE3),which is induced by 1mM IPTG at 20℃for 6 hours.After purification, we can get the purified recombinant GST-ICAM-1.SDS-PAGE reveald the two straps which is respectively 38.9kD for ICAM-1 extracellular domainⅠ～Ⅲand 27.9kD for GST by the contribution of enzymolysis of thrombin 60 hours at 4℃.We got about 6mg of purified rabbit anti-human ICAM-1 IgG from 3ml of rabbit anti-human ICAM-1 serum by the methods of saturated ammonium sulfate and the Protein A Sepharose CL-4B resin.After that we can get the purified and has immunological activity protein of ICAM-1 by CNBr-activited Sepharose CL-4B gel immunoaffinity chromatography.2.The Expression Separation and Purification of Extracellular domainⅠ～Ⅲof GST-ICAM-1 by HPLCWe determined the mobile phase of HPLC was 3%acetonitrile,0.02%TFA, 0.02Mammonium acetate,PH6.0 by repeatedly research.We can get the protein of immunological activity which is identified by Western Blot,but the effect is not satisfied because there are two straps on SDS-PAGE.3.The expression purification appraisement of the fusion protein of extracellular domainⅠ～Ⅲof His-ICAM-1.The recombinant plasmid of ZpET28aICAM was transformed into E.Coli BL21(DE3) which was renew constructed,induced by 1mM IPTG at 20℃for 6 hours.The production rate of His-ICAM-1 was 4.0mg/L LB media after purification.Conclusion:1.The protein of ICAM-1 extracellular domainⅠ～Ⅲwhich has immunological activity can be successfully separated from mixture by antibody mediated immunoaffinity chromatography.2.The protein which has immunological activity by HPLC in separating ICAM-1 extracellular domainⅠ～Ⅲand GST,but the effect is not satisfied because there are two straps on SDS-PAGE.3.The recombinant plasmid PET28aICAM-1 is successfully constructed which has only His.tag after purified by Ni-NTA affinity chromatograph.This can reduce the step and cost of purification.