绿脓杆菌外毒素A受体结合亚基-结核分枝杆菌热休克蛋白65融合蛋白的表达及纯化  

Expression and purification of fusion protein of pseudomonas exotoxin A receptor binding subunit and Mycobacterium tuberculosis heat shock protein 65

作　　者：冯越[1,2,3] 张国利[2,3] 赵福广[1] 万忠海[2] 岳玉环[2,3] 吴广谋[2,3] 田园[2,3] 刘雪[1,2,3] 张亮[1,2,3] 朱平[2,3] 

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林省人兽共患病预防与控制重点实验室省部共建重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2013年第10期1395-1399,共5页Chinese Journal of Biologicals

摘　　要：目的在大肠埃希菌中融合表达绿脓杆菌外毒素A(pseudomonas exotoxin A,PEA)受体结合亚基-结核分枝杆菌热休克蛋白65(heat shock protein 65,HSP65),并进行纯化。方法从含PEA的质粒中扩增PEA受体结合区亚基PEAⅠ,克隆至pET-28a-HSP65质粒中,构建重组表达质粒pET-28a-HSP65-PEAⅠ,转化大肠埃希菌(E.coil)BL21(DE3),IPTG诱导表达,表达的重组融合蛋白经SDS-PAGE分析;利用抗PEA受体结合区单抗杂交瘤细胞制备抗PEA受体结合亚基单抗,偶联免疫亲和层析介质,将表达的重组蛋白经DEAE Sepharose 4FF阴离子交换层析、Phenyl Sepharose 6FF疏水层析和免疫亲和层析进行纯化。结果重组质粒经PCR鉴定及测序,证明构建正确;表达的重组HSP65-PEAⅠ蛋白相对分子质量约93 000,主要以可溶性形式表达,表达量占菌体总蛋白的30%以上;抗PEA受体结合亚基单抗效价可达1∶10 000。纯化的重组HSP65-PEAⅠ蛋白纯度达90%以上。结论已成功在大肠埃希菌中表达并纯化了重组融合蛋白HSP65-PEAⅠ,为防止PA感染后多器官衰竭综合征的免疫制剂的研究奠定了基础。Objective To express the fusion protein of pseudomonas exotoxin A receptor binding subunit and Mycobac- terium tuberculosis heat shock protein 65 （HSP65-PEA I ） in E. coli and purify the expressed product. Methods The PEA I gene encoding PEA receptor binding subunit was amplified from PEA-containing plasmid by PCR and cloned into vector pET-28a-HSP65. The constructed recombinant plasmid pET-28a-HSP65-PEA I was transformed to E. coil BL21 （DE3） for expression under induction of IPTG. The expressed recombinant fusion protein was identified by SDS-PAGE. The monoclonal antibody （McAb） against PEA receptor binding subunit was prepared by hybridoma technique and cou- pled to the immune affinity chromatography medium, based on which the expressed recombinant protein was purified by DEAE Sepharose 4FF anion exchange chromatography, Phenyl Sepharose 6FF hydrophobic chromatography and immune affinity chromatography. Results PCR and sequencing proved that recombinant plasmid pET-28a-HSP65-PEA [ was con- structed correctly. The expressed recombinant HSP65-PEA I protein, with a relative molecular mass of about 93 000, mainly existed in a soluble form and contained more than 30% of total somatic protein. The titer of prepared McAb against PEA receptor binding subunit reached 1 ： 10 000. The purity of purified recombinant HSP65-PEA I protein was more than 90%. Conclusion Recombinant HSP65-PEA I protein was successfully expressed in E. coil and purified, which laid a foundation of development of immune agents for prevention of multiple organ failure syndrome after PA infection.

关 键 词：绿脓杆菌外毒素A 分枝杆菌 热休克蛋白 融合蛋白 表达 纯化 

分 类 号：Q253[生物学—细胞生物学]