麦胚无细胞蛋白合成系统表达人成纤维细胞生长因子21蛋白的研究

Expression of human fibroblast growth factor 21 protein in wheat germ cell-free protein synthesis system

中文关键词:  小麦胚芽  无细胞蛋白合成  人成纤维细胞生长因子21  蛋白质纯化

英文关键词:wheat germ  cell-free protein synthesis  human fibroblast growth factor 21  protein purification

中文摘要:

      目的 通过构建麦胚无细胞蛋白合成系统(wheat germ cell-free protein synthesis system, WGCF)体外表达人成纤维细胞生长因子21 (fibroblast growth factor 21, FGF21)。方法 构建FGF21克隆载体, 对FGF21基因进行克隆, 提取DNA后通过聚合酶链式反应(polymerase chain reaction, PCR)扩大底物浓度并体外转录成mRNA。制备麦胚抽提物溶液, 加入反应底物、能源物质和相关酶系构建WGCF。以mRNA为模板利用WGCF体外合成FGF21重组蛋白, 利用酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)对纯化后的FGF21重组蛋白进行检验, 并计算WGCF合成FGF21重组蛋白的产量。结果 成功利用构建的WGCF表达了FGF21蛋白, 最终合成产量为12.49 ng/mL。从基因层面到体外表达FGF21重组蛋白仅需2~3 d。结论 以麦胚抽提物为核心的WGCF可以正确合成具有复杂结构的真核蛋白, 为小麦制粉副产物麦胚的深层次开发利用提供有效手段和研究方向。

英文摘要:

      Objective To establish a wheat germ cell-free protein synthesis system (WGCF) and express human fibroblast growth factor 21 (FGF21) in vitro. Methods FGF21 cloning vector was constructed to clone the FGF21 gene. After DNA was extracted, the substrate concentration was amplified by polymerase chain reaction (PCR) and the gene was transcribed into mRNA in vitro. The wheat germ extract solution was prepared, and WGCF was constructed by adding reaction substrates, energy substances, and related enzymes. FGF21 recombinant protein was synthesized by WGCF using mRNA as a template in vitro. The purified FGF21 protein was detected by ELISA kit, and the yield of FGF21 recombinant protein synthesized by WGCF was calculated. Results FGF21 protein was successfully expressed in the constructed WGCF, and the final yield was 12.49 ng/mL. It only took 2-3 days from gene level to the expression of FGF21 recombinant protein in vitro. Conclusion WGCF can correctly synthesize eukaryotic protein with complex structure, which provides an effective means and research direction for further development and utilization of wheat germ, a by-product of wheat milling.