噬菌体展示技术筛选与白介素-32相互作用的蛋白  

Identification of proteins interacting with interleukin-32 via T7-phage display screening system

作　　者：孙伟[1] 庄重[1] 冯金荣[1] 孙晓雷[1] 朱丹丹[1] 段义农[1] 

机构地区：[1]南通大学医学院病原生物学系,226001

出　　处：《免疫学杂志》2013年第12期1038-1042,共5页Immunological Journal

基　　金：国家自然科学基金(81171589);江苏省高校自然科学基金(11KJB310008);南通市科技计划项目(K2010035);南通大学自然科学预研项目(10ZY009)

摘　　要：目的利用T7噬菌体展示技术筛选与白介素-32(interleukin-32,IL-32)相互作用的蛋白。方法构建IL-32α的原核表达重组质粒,表达并纯化重组IL-32α蛋白(recombinant IL-32α,rIL-32α);以纯化后的rIL-32α作为靶蛋白,应用T7噬菌体展示技术对人肝细胞cDNA文库进行筛选;对筛选到的阳性克隆进行DNA测序及计算机辅助分析;利用哺乳动物双杂交实验和免疫共沉淀实验对筛选到的阳性克隆进行验证。结果成功表达并亲和纯化了rIL-32α蛋白;经过5轮筛选,投入和产出的噬菌体滴度比值达到稳定;同源性分析初步确定9个与IL-32α蛋白相互作用的蛋白;进一步实验证实IL-32α与这9个蛋白片段能发生相互作用。结论利用T7噬菌体展示技术成功筛选到9个与IL-32α相互作用的蛋白,我们的发现为深入揭示IL-32的生物学功能奠定了研究基础。Interleukin-32（IL-32） is a recently-described proinflammatory cytokine. In this study, we attempt to identify proteins that interact with IL-32. Firstly, a recombinant plasmid of pET28a-IL-32αwas constructed and then introduced into E. coli BL21 （DE3） to express recombinant IL-32α （rlL-32α）. SDS-PAGE analysis showed that rlL-32a was mainly expressed in the periplasm of host bacteria as soluble form. Then the rlL-32α was purified by His-bind resin column. Using rlL-32α as target protein, we performed the T7 phage display assay for 5 rounds from human liver cDNA library. The phage plaques were detected by PCR, and the PCR products were then subjected to DNA sequencing. Computer-assisted analysis of DNA sequencing results revealed that 9 protein peptides might interact with rIL-32α. Furthermore, mammalian two-hybrid assays and co-immunoprecipitation assays confirmed the interaction between the 9 proteins peptides and IL-32α. Taken together, we successfully identify 9 proteins that interact with IL-32. Our finding provides a novel insight for fully understanding the role of IL-32.

关 键 词：白介素-32 T7噬菌体 原核表达 哺乳动物双杂交实验 

分 类 号：Q78[生物学—分子生物学]