Expression Of Recombinant Human Heparanase Protein And Preparation Of Its Monoclonal Antibody

Background and ObjectiveCancer, one of the most important diseases, has serious impacts on human health at present. The tumor invasion and metastasis is the main reason leading to the death of cancer patients. Metastasis process is very complex, now that the metastatic cancer cell invasion and metastasis, mainly through the following three steps: 1. tumor cell through cell surface receptors and cell adhesion to extracellular matrix protein first with specificity, 2. extracellular matrix hydrolase degradation, 3. degradation occurred tumor cell migration and invasion. And the factors involved in extracellular matrix (ECM) in the degradation process have attracted increasing attention.Heparanase in 1999 by four different laboratories successfully cloned a tumor metastasis-related genes, heparanase (Hpa) is the only way to cracking the major ECM components within the proteoglycan heparan sulfate proteoglycan (HSPG) of endogenous glucosidase enzyme. Studies show that the degradation of HSPG by heparanase way undermines ECM, BM integrity and the promotion of tumor angiogenesis and metastasis. More and more studies confirm that in almost all of heparanase in the expression of both advanced cancer and poor prognosis of cancer patients, and inhibition of heparanase expression can significantly reduce tumor invasion and metastasis. Thus, heparanase may serve as cancer diagnosis and therapeutic target.Monoclonal antibody (Monoclonal Antibody, McAb) is a single B lymphocyte clone secreting antibodies. Each B lymphocyte has synthesized a kind of antibody genes. When the body is stimulated by the antigen, the antigen molecules were many determinants each with different genes activated B cells. Activated B cell was proliferation of the cells in the formation of children, which is called cloning. If the election of a synthesis of an antibody to the cell culture, can be obtained from the single cell formed by the proliferation of the cell groups, which is called monoclonal. In 1975, Kohler and Milstein first reported that by the sheep red blood cells (SRBC) immunization of mouse spleen cells with mouse myeloma cell fusion, creating the first B cell hybridoma cell lines obtained anti-SRBC monoclonal antibodies. This is immunology, and even a milestone in the history of medicine. Monoclonal antibody is characterized by: a high degree of homogeneous physical and chemical properties, biological activity of single, antigen binding specificity, and easy handling and quality control people. These advantages make it come out to be a high priority, and the solution to biology and medicine, an important means many important issues. Monoclonal antibodies are highly specific, high purity characteristics, so that within a large concentration of low background, is particularly useful in immunohistochemistry, immunoprecipitation and immunoblotting, and have high specificity, the advantages of low background. Another monoclonal antibody can also be used for immunoaffinity purified antigen; the monoclonal antibody markers can also be used for rapid diagnosis and vaccine research. At present, the heparanase monoclonal antibodies prevalent in the foreign company, there is no commercialization of our heparanase monoclonal antibodies. The monoclonal antibodies imported from abroad are expensive and high cost.Based on the above points, this study was to build effective human heparanase gene expression, to obtain recombinant human heparanase, and then use access to high-purity protein as immunogen heparanase, routine immunization procedures and the hybridization technique to screen a human heparanase-specific monoclonal antibody. Immunohisto- chemistry, ELISA and Western Blot techniques were used on the available human heparanase monoclonal antibodies, and finally get specific human heparanase monoclonal antibody, for the future of heparanase monoclonal antibody in clinical diagnosis and antibody-mediated tumor targeted therapy to provide experimental basis.Methods1. The human heparanase full cDNA was cloned into pET30a plasmid, and further transformed into host strain BL21 (DE3) in prokaryotic expression. The human heparanase recombinant protein was purified by Ni2+ column purification, and expression of human heparanase recombinant protein was analyzed by mass spectrometry.2. Heparanase protein was used to immune Balb / c mice, producing antibodies of the B lymphocyte heparanase. Hybridoma cell lines secreting heparanase antibody were prepared; by limiting dilution screening of antibodies was strongly positive clone, and supernatant ELISA, positive identification of selected clones.3. HIS affinity chromatography purification and refolding purification was used to purify heparanase monoclonal antibody. Antibody obtained was used Western Blot technique, immunohistochemistry and ELISA to detect heparanase preparation of monoclonal antibody specificity and the efficiency of antibody price.Results1. We used PCR cloning of human heparanase protein coding genes. The sequence of the gene sequence with GENBANK provides exactly the same. The cloning of human heparanase gene was cloned into the prokaryotic expression vector pET30a heparin was constructed recombinant prokaryotic expression plasmid; by 1.0mmol / L IPTG 37℃induced 5h, pET30a-Hpa expression of fusion protein in BL21, a molecular weight of about 63KD, total bacterial protein expression more than 30%. Protein was identified after breaking the main bacteria in the deposition, expressed as inclusion bodies. Purified recombinant human heparanase protein was obtained by HIS affinity chromatography and refolding purification.2. Purified human heparanase protein immunogen was using conventional immunization procedures and hybridoma techniques, by cell fusion, screening out 15 specific anti-human heparanase monoclonal antibodies, identified by the sub-class of which 3 strains suitable for use in the diagnosis reagent IgG1.3. By immunohistochemistry, ELISA, and Western to get the heparin on the enzyme specificity of monoclonal antibodies and titers of identification, the results show that the prepared 3 heparanase specific antibody, and the high titer monoclonal antibodies.Conclusion1. We used PCR cloning of human heparanase protein coding genes. High-purity human heparanase protein was obtained by the identification of the heparanase protein molecular weight determination consistent with the theoretical molecular weight, which can be used for heparanase monoclonal antibodies.2. We have successfully prepared three specific human heparanase monoclonal antibodies. These three human heparanase monoclonal antibodies were identified by ELISA, Western Blot and immunohistochemistry, confirming the high specificity and high titer. The study suggests that our preparation of heparanase monoclonal antibodies may be used to heparanase as a target for cancer diagnosis and treatment.