Expression And Purification Of Heparanase In Prokaryotes And The Application Of Its Antibody In Clinical Analysis

Heparanase (HPA) is the key enzyme in the process of tumor metastasis through specific identification and degradation of heparin sulfate (HS) side chain of heparan sulfate proteoglycan (HSPG). Thus, it not only causes the destroy of extracellular matrix (ECM) and the structural integrity of the basement membrane (BM) blood vessels, but also make the release of growth factors, chemotactic factors and bioactive molecules combined with side chain of HS, which play important roles in the process of tumor growth, transfer, new blood vessels formation. In the mid-1980s, it was the first time discovered that tumor metastasis are closely related to heparanase. Then, many scientists were devoted to the study. However, because the content of heparanase is low, the property is unstable and difficult to separate, rapid, accurate and facilitate the detection methods are lack. Therefore, these are seriously hindered the research and application of heparanase in clinical. During late 1990s, complete heparanase gene had been successfully cloned, which made great contribution to the research. It was proved that almost all tumors (including leukemia) occur accompanied with high expression level of heparanase and activity, especially when abnormally high level of heparanase. The experiments indicated that heparanase is closely related to the properties of tumor and prognosis. Hence, the method of detecting human sera heparanase is very important in tumor early diagnosis and the risk assessment of metastasis, especially in cancer screening test and clinical diagnosis together with some other tumor markers.So far, high purity and enough heparanases could not be obtained from cells, tissues and body fluids through protein purification because the degradation and purification of heparanase are influenced by many factors. So, the most ideal choice is using gene recombination technique to obtain heparanase and its antibody by immunization technique. The cDNA of truncated human heparanase (without signal peptide) was cloned by PCR using pcDNA3.1-HPA (constructed by our lab) as template, and subcloned into pET-28a (+) vector. Then the expression plasmid was transformed into E.coli BL21 (PlyS, DE3). Following recombinant human heparanase was obtained by optimizing the initial induction density, induction time, temperature and concentration of inducer. Then, the target protein was purified by using HisTrapTMcrude affinity chromatography. The purity of the recombinant of heparanase was confirmed by SDS-PAGE and Western blotting. The results indicated that high purity and enough heparanase was obtained and couled be used to prepare anti-heparanase antibody.At present, there are many studies on mechanism of heparanase and tumor, in which the mainly detection method is immunohistochemistry, quantitative RT-PCR or semi-quantitative RT-PCR. Due to lack commercial standard sample of heparanase and strong specificity of anti-heparanase, quantitative detection method is not established in home and abroad. Therefore, to prepare strong specificity of anti-heparanase, 6-8 weeks of female BALB/c, mice were subcutaneous immunized by the recombinant enzyme every three weeks. A single dose of immunizaion was 30μg, following 4 boosting immunization. Then the mouse with high titer sera was selected, separated splenic cells and fused with SP2/0 myeloma cells to prepare hybridoma cells. Positive clones were screened a week later by ELISA. Then, positive subclones were obtained by limiting dilution through three subcloning and monoclones were picked. And 8-10 weeks female BALB/c mice were injected liquid paraffin. 10 days later the mice were injected hybridoma cells. And one week later when the hybridoma cells in mice were enough, ascites were extracted and centrifugated to collect supernatant. And the ascites were purified by HiTrap rProteinG chromatography and the titer was determined by ELISA. Based on the specificity and affinity of the antibody secreted by hybridoma cells, the results showed that the anti-heparanase monoclonal antibody was obtained, which could be used in detecting heparanase levels in sera.Compared with immunohistochemistry and in-situ PCR detection methods, the ELISA method has many advantages, such as convenient materials acquirability, easy and simple operation, accurate quantification and less interference factors. So, the method is suitable for popularization and use. Through adopting double sandwich ELISA method, both manual and automated instrument can also be completed. Accompanied with regular detection of tumor markers, it is more advantageous to screen cancer and diagnosis. However, because the content of heparanase in sera is very low, the antibody with strong specificity and high titer is becoming particularly important. At present, there is no heparanase assay kit in home and abroad and the price of heparanase monoclonal antibody is very expensive. Considering cost factors and the antibody specificity, we chose M05 monoclonal antibodies (Abnova company, mouse) and PP1320 polyclonal antibody (Acris company, rabbit) as controls of our monoclonal antibody to detect the levels of heparanase in lung cancer (25 cases), gastrointestinal tumors (42 patients) and normal sera. The result showed that the method was simple, stable and repeatable. Moreover, the result found that the enzyme in cancer patients sera was significantly higher than normal, which was consistent with the result of immunohistochemistry and RT-PCR reported by abroad. Furthermore, the method of sera detection is a noninvasive detection and can be used to diagnosis and prognosis of cancer development.To sum up, in this study, we constructed procaryotic expression vector of human heparanase pET 28a (+)/HPSE. And the expression of the target protein was used as immnogen to produce anti-heparanase monoclonal antibody. The result indicated that we obtained high specific antibody, which could be used in detecting heparanase levels in sera.. Based on the above results, we developed a simple, stable and repeatable method, double sandwich ELISA, to detect heparanase levels in sera. Through detecting the heparanases in cancers and normal sera, the result suggested that the method is feasible and has clinical significance.