毕赤酵母表达的HIV-2外膜糖蛋白的纯化与活性测定

人免疫缺陷病毒(Human immunodeficiency virus,HIV)是艾滋病（AIDS）的病原体，其外膜蛋白是HIV侵入人体靶细胞的物质基础，对其结构与功能的深入研究不但能为彻底揭示HIV致病机理奠定基础，而且有助于抗感染免疫研究和治疗物的设计^1,2]，HIV－2ROD外膜蛋白gp105是一种高度糖基化的糖蛋白，近年来发展迅速的毕赤酵母（Pichia Pastoris）真核表达系统对分泌蛋白的糖基化修饰形式与天然的gp105相同^3]。为实现大量制备有活性的gp105，我们用PCR法去除了gp105基因5′端28个密码子的非功能片段，以提高gp105的表达水平，并成功地在毕赤酵母中实现了gp105的高效分泌表达。本文对这一表面产物的纯化、性质及活性进行了研究，为尽快研制出有效的疫苗奠定基础。

Purification and Characterization of Human Immunodeficiency virus Type 2 External Glycoprotein Expressed in Pichia Pastoris

Expression conditions of human immunodeficiency virus type 2 external glycoprotein gp105 in the recombinant Pichia Pastoris strain were optimized via orthogonal test of some factors such as the rate of aeration, the inductive duration, the initial pH and the concentration of methanol. The results from tests of between subjects effects showed that the most important parameter for efficient expression of gp105 in recombinant Pichia Pastoris strain is adequate aeration during methanol induction, and the optimum inductive condition for gp105 expression was: more than 80% aeration, 3 days for induction, th einitial pH of 6 0-7 0, the final methanol concentration of 1 0%-1 5%. With this condition, the expressed gp105 was secreted into fermentation broth and reached a ield of 30%, approximately 200 mg/L. Expressed gp105 was isolated and purified by sating out and Sephadex G 100 chromatography and the yield of gp105 was 40%. gp105 was purified to electrophoretic purity and its pI was about 5 0 by SDS PAGE and isoelectrofocusing. Its N terminal amino acid was arginine by Dansyl Cl and the result indicated that expressed gp105 was secreted and cleavaged correctly. The results from ELISA demonstrated that the purifiec gp105 showed good reactiongenicity and antigenic specificity.