抑制HIV包膜蛋白亚基gp41六股螺旋形成的链霉菌多糖的分离与结构分析

目的从链霉菌发酵液中定向分离纯化抗HIV1包膜糖蛋白gp41六股α螺旋束形成作用的多糖成分,并进行结构分析。方法乙醇沉淀多糖类大分子物质、Sevage法去蛋白、DEAE纤维素离子交换层析法及SephadexG25凝胶过滤法纯化多糖。其结构经HPLC、UV、IR、1HNMR以及高碘酸氧化、Smith降解等方法进行测定。抗HIVgp41活性检测采用夹心ELISA方法。结果分离纯化出的目的多糖属于中性多糖,分子量约为4855ku;单糖组成以葡萄糖为主,含有少量果糖,二者的摩尔比约为22∶1(1096∶048);糖苷键的链接方式为(1→4)αD吡喃型,并含1→6位键合的支链;命名为SMP。SMP对HIVgp41六股α螺旋束的形成有明显抑制作用,其半效抑制浓度(IC50)为(14548±725)mg·L-1。结论从链霉菌发酵液中分离纯化出一种新的抑制HIV包膜糖蛋白gp41六股α螺旋束形成的链霉菌多糖。

Isolation and structure analysis of streptomyces polysaccharide with inhibit HIV envelope glycoprotein subunit gp41 activity

Aim To isolate, purify, and chemically characterize a polysaccharide showing inhibition of the six-α-helix bundle formation of HIV-1 envelope glycoprotein gp41 from cultured broth of a streptomyces sp.strain. Methods Ethanol was used to precipitate polysaccharides and macromolecules from the broth.Proteins in the precipitate were removed by sevage method.Purification was carried out by DEAE-Cellulose and sephadex G-25 column chromatography. The chemical structure of the polysaccharide was determined with the combined application of HPLC,UV,IR and 1H-NMR spectroscopy, and the methods of periodate oxidation and Smith degradation,etc. Activity of anti-HIV-1 ~gp41 six-α-helix bundle formation was assayed withsandwich ELISA method.Results The purified polysaccharide,designated as SMP for Streptomyces polysaccharide,is neutral with a molecular weight of approximately 4855 Daltons. Sugar analysis showed SMP contains glucose and fructose residues in an approximate molar ratio of 22∶1 (10.96 to 0.48). The glycosidic linkages were estimated to be (1→4)-α-D-pyranoside as its main chain, and 1→6 linkage was attached to the main chain. Activity analysis revealed SMP markedly inhibited the six-α-helix bundle formation of HIV-1 glycoprotein gp41 and the IC_~50 was (145.48±7.25) mg·L~-1 .Conclusion Streptomyces polysaccharide SMP showing inhibition of the six-α-helix bundle formation of HIV-1 envelope glycoprotein gp41 was isolated and purified.