人源抗HBsAg单链抗体与白细胞介素2融合蛋白在巴氏毕赤酵母中的表达

目的：探讨抗HBsAg单链抗体与白细胞介素2融合蛋白在巴氏毕赤酵母(P．Pastoris)中的表达以及初步的纯化、活性鉴定方法。方法：用PCR将目的蛋白基因从质粒PGEM7Zf( )-HBScFv-IL-2上扩增出来，再亚克隆到酵母表达载体pPICZaA中，转化P．Pastoris宿主菌GS 115，菌落：PCR、高浓度Zeocin抗性筛选鉴定转化子，重组酵母经甲醇诱导后，通过SDS-PAGE及Western-blot分析鉴定表达产物；再通过亲和层析法纯化目的蛋白；用间接ELISA实验鉴定其活性。结果：表达的重组蛋白分子质量约为44ku，表达量可达12％；纯化后凝胶成像分析目的蛋白的纯度达到95％；重组融合蛋白能与HBsAg、鼠抗IL-2单克隆抗体特异性结合。结论：成功构建了抗HBsAg单链抗体与IL-2融合蛋白酵母表达工程菌，并初步建立了对目的蛋白的纯化及活性鉴定方法。

The Expression of A Recombinant Fusion Protein Consisting of Anti-HBsAg Single Chain Fv and Interleukin-2 in P.Pastoris

Objective:To explore the expression of a recombinant fusion protein of Anti-HBsAg single chain Fv and interleukin-2 in P.Pastoris, and the primary method of its purification and evaluation.Methods:The target protein (HBScFv-IL-2) gene was amplified from plasmid PGEM7Zf ( ) -HBScFv-IL-2, and was subcloned to the yeast expression vector pPICZaA.The resultant plasmid pPICZa-HBScFv-IL-2 was linearized and transformed into P.Pastoris GS115.The recombinant Pichia strains were identified by PCR and zeocin-resistant screening of Pichia transformants were cultured and induced with methanol. The expression production was detected by SDS-PAGE and Western-blot and purified by affinity chromatography.The bioactivity of purified product was detected by indirect ELISA. Results:The target protein with 44 ku molecular weight could reach 12%.The purity of target protein could reach 95% after purification. The recombinant fusion protein could be specifically connected with HBsAg and mouse anti-IL-2.Conclusion:An Pastoris engineering bacterium for HBScFv and IL-2 fusion protein is constructed and the methods for the purification and bioactivity assay of target protein are established.