摘要
目的克隆表达C9orf47-1蛋白,探索其可能的生物学功能。方法构建C9orf47-1原核表达载体,转化BL21,IPTG诱导目的蛋白表达;纯化目的蛋白,制备兔源多克隆抗体;采用生物信息学软件对C9orf47-1蛋白进行生物信息学分析;采用Western blot检测其在HepG2、L02、LX2和Jurkat细胞系的表达情况,及重组蛋白对HepG2细胞内质网应激的影响;采用流式细胞术检测其对HepG2细胞周期的影响。结果 PCR扩增获得序列完全正确的C9orf47-1基因片段,构建的C9orf47-1原核表达载体能在大肠埃希菌中以包涵体的形式表达C9orf47-1重组蛋白;重组蛋白经Ni-NTA纯化后免疫大耳白兔,获得的多克隆抗体效价高达1∶640 000,并识别C9orf47-1蛋白;C9orf47-1蛋白在4种细胞系中均有表达,其中在HepG2和Jurkat中的表达量高于L02和LX2;重组蛋白未引起HepG2细胞的内质网应激,但可引起细胞周期的变化:延长G1、G2期,缩短S期(P<0.05)。结论 C9orf47-1重组蛋白具有抗原性,该蛋白对肝细胞周期(G2/S)有一定的阻滞作用。
Objective To clone and express the recombinant protein C9orf47-1and to investigate its potential biological functions. Methods The C9orf47-1gene was inserted into a prokaryotic expression plasmid and transformed into E.coli BL21.Protein expression was induced with IPTG and the resulting protein was purified using Ni-NTA.Specific polyclonal antibodies were obtained from immunized rabbits.The recombinant protein C9orf47-1was bioinformatically analyzed using predictive software.Western blotting was used to observe the expression of C9orf47-1protein in HepG2,L02,LX2,and Jurkat cells.The impact of the recombinant protein C9orf47-1on endoplasmic reticulum stress（ERS）was determined using Western blotting and its impact on the cell cycle of HepG2cells was determined using flow cytometry. Results A correctly sequenced C9orf47-1gene was obtained,and the recombinant protein C9orf47-1was highly expressed by E.coli in the form of inclusion bodies.Purified recombinant protein was obtained using Ni-NTA resin and was used to immunize rabbits.The titer of polyclonal antibody peaked at 1：640,000.Antibodies were specific for the recombinant protein C9orf47-1.C9orf47-1protein was noted in all four of the cell lines tested,and it was expressed at a higher level in HepG2and Jurkat cells than in L02and LX2cells.Recombinant protein C9orf47-1had no impact on ERS in HepG2cells.However,it regulated the cell cycle of HepG2cells（P〈0.05）by prolonging the G1and G2phases and shortening the S phase（P〈0.05）. Conclusion The recombinant protein C9orf47-1is antigenic,and it has a certain level of action to block the cell cycle（the G2and S phases）of liver cells.
出处
《中国病原生物学杂志》
CSCD
北大核心
2014年第4期364-368,371,共6页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.81071411
81271901)
