病毒巨噬细胞炎性蛋白-Ⅱ在毕赤酵母中的表达及其抗HIV-1活性  

Expression of Viral Macrophage Inflammatory Protein-Ⅱin Pichia pastoris and Its Anti-HIV-1 Activity

作　　者：莫雪梅[1] 孙晗笑[1] 贾忠伟[1] 李秀英[1] 张光[1] 

机构地区：[1]暨南大学药学院基因组药物研究所,广州510632

出　　处：《中国生物制品学杂志》2011年第6期629-633,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(308272221);2011年暨南大学青年教师科研培育与创新基金(中央高校基本科研业务费专项资金)

摘　　要：目的在毕赤酵母中表达病毒巨噬细胞炎性蛋白-Ⅱ(Viral macrophage inflammatory protein-Ⅱ,vMIP-Ⅱ),并检测其抗HIV-1活性。方法通过PCR技术从质粒pQE-vMIP-Ⅱ中扩增vMIP-Ⅱ基因,插入酵母表达载体pPICZaA中,构建表达质粒pPICZaA-vMIP-Ⅱ,转化毕赤酵母菌X33,甲醇诱导表达。表达产物经镍离子亲和层析和分子筛层析纯化后,进行Western blot鉴定,并检测重组vMIP-Ⅱ蛋白对HIV-1诱导的细胞合胞体形成的抑制作用。结果重组表达质粒pPICZaA-vMIP-Ⅱ经酶切及测序鉴定证明构建正确;表达的重组vMIP-Ⅱ蛋白的相对分子质量约为8 500,以分泌形式表达于重组酵母菌的发酵上清中;纯化的重组蛋白纯度达97.3%,且可与HRP标记的vMIP-Ⅱ多克隆抗体发生反应;重组vMIP-Ⅱ蛋白组的合胞体数目明显少于阳性对照组(P﹤0.05),其IC50值为1.35 ng/ml。结论已成功在毕赤酵母中表达了重组vMIP-Ⅱ蛋白,其具有较强的抑制HIV-1的活性。Objective To express viral macrophage inflammatory protein-Ⅱ（vMIP-Ⅱ） in Pichia pastoris and determine its anti-HIV-1 activity.Methods The vMIP-Ⅱ gene was amplified from plasmid pQE-vMIP-Ⅱ by PCR and inserted into expression vector pPICZaA.The constructed recombinant plasmid pPICZaA-vMIP-Ⅱ was transformed to P.pastoris X33 for expression under induction of methanol.The expressed product was purified by nickel ion affinity chromatography and size-exclusion chromatography,identified by Western blot and determined for inhibitory effect on cell syncytium formation induced by HIV-1.Results Both restriction analysis and sequencing proved that recombinant plasmid pPICZaA-vMIP-Ⅱ was constructed correctly.The expressed recombinant vMIP-Ⅱ protein,with a relative molecular mass of about 8 500,existed in a secretory form in fermentation supernatant,reached a purity of 97.3% after purification and showed specific reaction with HRP-labeled polyclonal antibody against vMIP-Ⅱ.Western blot showed that recombinant vMIP-Ⅱ was expressed in secretory form in culture supernatant.The number of syncytia in MT4 cells treated with recombinant vMIP-Ⅱ was significantly smaller than that in positive control group（P 0.05）.The IC50 of recombinant vMIP-Ⅱ to syncytium formation was 1.35 ng/ml.Conclusion Recombinant vMIP-Ⅱ protein was successfully expressed in P.pastoris,which showed high activity in inhibiting HIV-1.

关 键 词：病毒巨噬细胞炎性蛋白-Ⅱ 毕赤酵母 HIV-1 

分 类 号：R373.9[医药卫生—病原生物学] Q786[医药卫生—基础医学]