重组人B7-H1IgV发酵纯化工艺的建立及其抑瘤活性

目的建立大规模生产重组人B7-H1IgV(rhB7-H1IgV)的发酵和纯化工艺,并检测纯化蛋白的抑瘤活性。方法对rhB7-H1IgV工程菌进行发酵培养,IPTG诱导表达,采用超声裂菌分离包涵体,梯度复性,2次Ni亲和层析等步骤纯化目的蛋白,并进行SDS-PAGE、Westernblot分析及抑瘤活性检测。结果在建立的发酵工艺下,rhB7-H1IgV的表达量可达菌体总蛋白的(10～11)%;纯化的rhB7-H1IgV蛋白经HPLC检测,纯度可达95%以上,Westernblot检测具有良好的反应原性;动物实验表明纯化的重组蛋白能诱导BALB/c小鼠产生高滴度的抗B7-H1抗体,且对肿瘤的生长具有明显的抑制作用。结论所建立的rhB7-H1IgV发酵纯化工艺简单迅速,为其开发和应用奠定了基础。

Development of Procedure for Fermentation and Purification of Recombinant Human B7-H1IgV and Antitumor Activity of Purified Protein

Objective To develop the fermentation and purification procedures for large-scale production of recombinant human B7-H1IgV(rhB7-H1IgV)and determine the antitumor activity of purified protein.Methods Recombinant E.coli DH5α with B7-H1IgV gene was cultured by fermentation and induced with IPTG for expression,then cleaved by ultrasonication,from which inclusion bodies were separated for gradient re-naturalization,and the target protein was purified by nickel ion affinity chromatography for 2 times,then identified by SDS-PAGE and Western blot,and determined for antitumor activity.Results The expressed rhB7H1IgV in E.coli DH5α cultured by the developed fermentation procedure contained(10 ～ 11)% of total somatic protein.The expressed rhB7-H1IgV reached a HPLC purity of more than 95% after purification,and showed good reactogenicity as proved by Western blot.The purified rhB7-H1IgV induced high anti-B7-H1 titer in BALB /c mice,and showed significantly inhibitory effect on growth of tumors.Conclusion The developed procedures for fermentation and purification was simple and rapid,which laid a foundation of further development and application of purified rhB7-H1IgV.