Cloning Of Human Endostatin Gene And Its Recombinant Expression, Purification And Biological Activity Characterization

BackgroundEndometriosis is a common benign gynecologic disease affecting 15% of female in vegetation period and 35% to 50% of barren women. It often causes infertility, dysmenorrhea, dyspareunia and pelvic pain. The pathogenesis and pathogeny are not completely understood. The aim of clinical therapy is focused on alleviating pain, improving pregnancy rate and delaying recurrence to the greatest extent. Suppression of ovarian activity by hormonal therapy or operation is the main therapeutic interventions with severe adverse effects and a high relapse rate. Nowadays, more and more study results showed that angiogenesis plays a major role in the development and maintenance of endometriosis. Therefore, according to the theory of angiogenesis, investigating endometriosis' new treatment approach to reduce its relapse rate and lessen side effects has been becoming an urgent task for gynaecologists. Endostatin, the most potent endogenous angiogenic inhibitor, is widely investigated and applied in cancer therapy, while its application in the treatment of endometriosis is seldom reported. ObjectiveIn order to obtain Human Endostatin protein for animal test, we try to clone the Human Endostatin gene from fetal liver tissue, express the gene in Escherichia coli and obtain high purity of recombinant Human Endostatin protein with biological activity in this research. Methods and Results1. Endostatin gene was cloned by Reverse Transcription Polymerase ChainReaction from fetal liver and its sequence was validated. Compared with Human Endostatin gene in Genbank, the gene we obtained has a site mutant at the 471 base and mutates from G to A. It was a silent mutation and the coded amino acid is still arginine.2. The cDNA of Human Endostatin was cloned into non-fusion expression vector pBV220 and expressed successfully in E.coli DH 5a. After washing and denaturing-renaturing, the recombinant Human Endostatin protein was purified by SP Sepharos Fast Flow ion exchange chromatograph and phenyl Sepharose hydrophobic interaction chromatograph. The purity of Human Endostatin above 95% was obtained.3. The cDNA of Human Endostatin was cloned into fusion expression vector pTRX and expressed successfully in E.coli BL21 (DE3). After washing and denaturing-renaturing, the fusion protein Trx-endo was purified by Ni2+-Chelating Sepharose interaction chromatograph. Cut with EK enzyme, recombinant endostatin protein was purified further by Ni2+-Chelating Sepharose interaction chromatograph and SP Sepharos Fast Flow ion exchange chromatograph. The purity of Human Endostatin above 95% was also obtained.4. The activity of recombinant protein was analyzed by MTT method. Human Endostatin protein can inhibit proliferation of ECV 304 cell in vitro and inhibit effect-dose relation was observed. Apoptosis of endothelial cells was also observed by fluorescence method and it may be the reason of inhibiting proliferation of ECV 304 cell induced by endostatin protein.ConclusionHuman Endostatin gene was cloned and expressed in E.coli both as a non-fusion protein and as a fusion protein. High purity of recombinant endostatin protein was obtained and its biological activity was analyzed on cultured ECV 304 cell in vitro. Those experiment results lay a good foundation for endometriosis antiangiogenesis therapy with endostatin gene.