Expression Of Human Arginase ? Gene And Preparation Of Its Polyclonal Antibody

Arginase I(ARG I),as a key enzyme of urea circulation in the liver,has many important biological functions and is closely related to the occurrence and development of many diseases,such as cancer,hypertension,aging,ischemia-reperfusion and diabetes mellitus.Therefore,ARG I has become a hotspot in the research of metabolic function.Argimase I can hydrolyze L-arginnine into L-ornithine and urea,in which L-ornithime is further produced polyamine and proline metabolites,which affect cell growth and structural protein synthesis.In addition,argimase activity also exists in the vascular system.In the vascular system,arginase competes with nitric oxide synthase(NOS)for L-arginmne,which inhibits the synthesis of nitric oxide(NO),which may lead to dysfunction of vascular endothelial cells or smooth muscle cells.Recent studies have shown that arginase is highly expressed in many malignant tumors,especially as a specific marker of hepatocellular carcinoma,it also plays an important role in the diagnosis and treatment of hepatocellular carcinoma.New studies have also found that arginase I is expected to become a new therapeutic target,which can reverse the dysfunction of vascular endothelial cells and smooth muscle cells and prevent vascular diseases.Therefore,exploring the structure and function of arginase I is helpful to confirm the role of arginase I in the occurrence and development of related diseases.The preparation of antibodies that can effectively recognize arginase I in physiological and pathological conditions can help to further study the structure and function of ARG I.In this study,the expression of human arginase I gene and the preparation of its polyclonal antibody were carried out,and the similarities and differences of protein immunity,DNA immunity and DNA primordial-protein enhanced immunity in the preparation of antibody were compared and analyzed,and a technical system for the effective preparation of human arginase I polyclonal antibody was explored.In order to prepare antibodies of human arginase I(human-ARG I),the first step is to clone human arginase I gene and to construct recombinant expression plasmids,further to prapare plasmid antigen and protein antigen that for animals immunization.Tree pairs of specific primers were designed according to the sequence of human arginase I gene provided by NCBI database.The target gene was amplified by PCR and pCDNA3.0 Flag-Arginase I as templates.The human arginase I gene was linked to pCMV-MYC,pEGX-4T-land pED-1 plasmid vectors respectively.The eukaryotic expression vector pCMV-MYC-ARGI,prokaryotic expression vector pEGX-4T-1-ARGI and pED-1-ARGI were constructed,and identified by restriction enzyme digestion and DNA sequencing.In order to obtain the protein antigen-human arginase I,prokaryotic expression vectors pEGX-4T-1-ARG I and pEDl-ARG I were inducing expression in competent cells of E.coli respectively,and the fusion proteins including arginase I with different labels(GST-ARGI,His-ARG I)were obtained.The specificity of the fusion protein was analyzed and selected the GST-ARGI to animals immunization.The results showed that the fusion proteins GST-ARGI(about 64 kDa)and His-ARGI(about 36 kDa)could be induced by prokaryotic expression vectors pEGX-4T-l-ARGI and pED1-ARGI.The optimal conditions for inducing expression were identified as 25 C,0.6 mmol L-1 isopropylthio-beta-D-galactoside(IPTG)and 16 h.The purity of the fusion protein could reach more than 90%by affinity purification.The fusion protein could specifically bind to GST-ARG I antiserum,and a specific binding band appeared at 36kDa.The obtained human arginase I protein can be used as an antigen to animals immunization.To obtain the polyclonal antibodies can effective recognize the human arginase I and to compare the similarities and differences between the different immune strategies.Animals were immunized according to the strategy of DNA immunization,protein immunization and DNA immunization-protein immunization.After the third immunization,the sera of each group were collected and their titers were tested.The sera of mice in protein immunization group and DNA immunization-protein enhancement group were purified.,and the titers and specificities of antibodies were further identified.In addition,the recombinant expression plasmid pCMV-MYC-ARGI was transfected into 293T cells to detect the expression of the target gene in mammalian cells.The results showed that the purity of the purified antibody was over 98%,the titer of the antibody was obviously improved,and the obtained antibody had high antigen specificity.The results of calcium phosphate cell transfection confirmed that the recombinant plasmid pCMV-MYC-ARGI could be highly expressed in mammalian cells and produce human arginase I protein with biological activity.The high titer antigen-specific polyclonal antibody against human arginase I not only establishes an efficient immune strategy for the preparation of antibodies,but also provides an effective detection tool for the in-depth study of the structure and function of arginase I.