| Angiogenin (Ang ) is a small monomer which universally exists in tissues and cells. Angiogenin belonging to ribonuclease superfamily has 35% homologous sequences with ribonuclease A(RNase A). Compared with ribonuclease A, the ribonuclease activity of Ang is very low, but is very important to promote new endothelia proliferation. The excessive vascularization is related to many pathologic processes. This phenomenon is much prominent during the growth of tumor. The growth and metastasis of tumor cell depend on formation of new vascular. Blood vessles and interstitium comprise 50 percent of net weight of tumor tissues. In addition to providing nutrition, vascular also supplies an access for tumor cells to go into circulatory system and metastasize. So, blockading the process of neovascularization is to prevent tumor from growing and metastasizing. Ang is involved in the pathologic processes of arthritis, scleroderma and diabetic retinopathy, too.Ribonuclease inhibitor (RI) is an acid cytoplasmic protein with the molecular weight of 50 kD. This ubiquitously distributed glycoprotein tightly binds and inhibits Ang with a stoichiometry of 1:1. The remarkable affinity of RI and Ang supplies an approach to invalid the function of Ang which is to stimulate neovascularization. The experiments had shown that inoculating RI into the transplanted tumor of mouse resulted in significant reduction of the amount of neovascular and massive necrosis of tumor cells. According to an important character of the primary structure of RI: 32 cysteine residues are all in the reduced form, we propose that RI can neutralize oxidization. This proposal was confirmed by the experiment that C6 cells transfected with ri could resist more injury of H2O2. In this sense, the production of RI with native function by genetic engineering will undoubtedly bring far-reaching effect.Our laboratory has carried out much work in the study of RI. In this experiment, ri gene was amplified by PCR method from recombinant cloning vector pT7-ri, which had been constructed by Dr. Yu Xiu-ping in this laboratory and sequenced by TaKaRaBiotechnology (Dalian) Co.,Ltd.. The 5' and 3' ends of the PCR product were respectively introduced into BamH I and EcoR I sites. Then, ligate ri fragment and the vector pGEX-2T to construct the fusion expression system pGEX-2T-ri. The ligating-product was transformed into competent E.coli DH5 a . The positive clone was screened and identified by PCR and analysis of restriction endonuclease digestion. After induced by 0.5 mmol/L IPTG for 4 hours, the expression amount of recombinant GST-RI is optimum, accounting for 15%~20% of total bacterial proteins. The recombinant protein with the molecular weight of 76 kD was mainly expressed as inclusion bodies in E.coli, which was identified by SDS-PAGE. After sonification, the precipitate was washed repeatedly by ionic and non-ionic detergents to remove membrane proteins as could as possible, and the cleaner inclusion bodies were abtained. Then the inclusion bodies were solubilized in 8 mol/L urea-100 mmol/L P -mercaptoethanol ( β -ME). The protein concentration was adjusted to 0.15 mg/ml, and the solution was dialysed against 2.5 mol/L urea- 100 mmol/L B -ME for 24 hours, making the recombinant protein to refold, correctly and completely. Finaly, the renaturant solution was dialysed against 20 mmol/L Tris-Cl (pH 7.5) to remove extra urea. After dialysis, the recombinant GST-RI was purified by RNase A-Sepharose 4B affinity chromatography, and the single band of GST-RI was showed on a SDS-electrophoresis gel to remove the extra urea. The elutes had the activity to inhibit RNase A ( up to 150 U/L). With the treatment of thrombin for 16 hours at 24℃, the fusion protein was cut into 26 kD GST and 50 kD RI. Western-blot identified that the crude inclusion bodies, the renatured GST-RI and thrombin-cleaved 50 kD RI had immuno-activity.Plasmid pGEX-2T is a fusion expression vector with a strong inducible promoter tac, lacIqgene and thrombin-recognizing site. It was because of lacI...
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