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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 the viral macrophage inflammatory protein-Ⅱ (vMIP-Ⅱ) in Pichia pastoris and test its anti-HIV-1 activity. Methods The vMIP-Ⅱ gene was amplified by PCR from plasmid pQE-vMIP-Ⅱ and inserted into yeast expression vector pPICZaA. The expression plasmid pPICZaA-vMIP-Ⅱ was constructed and transformed into Pichia pastoris X33. The expressed product was purified by nickel ion affinity chromatography and molecular sieve chromatography, and then identified by Western blot. The inhibitory effect of recombinant vMIP-Ⅱprotein on HIV-1-induced syncytia formation was detected. Results The recombinant plasmid pPICZaA-vMIP-Ⅱ was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant protein vMIP-Ⅱ was expressed in the secreted form of recombinant yeast vMIP-Ⅱ with a molecular weight of about 8 500. The purity of recombinant protein was 97.3%, and it could react with polyclonal antibody of vMIP-Ⅱ labeled with HRP. The number of syncytia in recombinant vMIP-Ⅱ protein group was significantly less than that in positive control group (P <0.05) ng / ml. Conclusion The recombinant vMIP-Ⅱ protein has been successfully expressed in Pichia pastoris, which has a strong inhibitory activity against HIV-1.