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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 C9orf47-1 protein and explore its possible biological functions. Methods The prokaryotic expression vector of C9orf47-1 was constructed and transformed into BL21. IPTG was used to induce the expression of the target protein. The target protein was purified to prepare polyclonal antibody. The bioinformatics software was used to analyze the C9orf47-1 protein. The protein was analyzed by Western blot In HepG2, L02, LX2 and Jurkat cell lines, and the effect of recombinant protein on the endoplasmic reticulum stress in HepG2 cells. The effect of recombinant protein on the HepG2 cell cycle was detected by flow cytometry. Results The complete C9orf47-1 gene fragment was obtained by PCR amplification. The constructed C9orf47-1 prokaryotic expression vector could express C9orf47-1 recombinant protein as inclusion body in Escherichia coli. The recombinant protein was purified by Ni-NTA Immunization of rabbits, the polyclonal antibody titers reached as high as 1: 64000, and C9orf47-1 protein; C9orf47-1 protein was expressed in all four cell lines, which in HepG2 and Jurkat high expression L02 and LX2. The recombinant protein did not induce endoplasmic reticulum stress in HepG2 cells, but could induce cell cycle changes: prolonging G1 and G2 phases and shortening S phase (P <0.05). Conclusion C9orf47-1 recombinant protein has antigenicity, and this protein has a certain retardation effect on the cell cycle (G2 / S).