尼罗罗非鱼补体3基因片段的原核表达及条件优化Prokaryotic Expression and Optimization of Complement 3 Gene Segment in Nile Tilapia(Oreochromis niloticus)
牛金中,黄瑜,汤菊芬,鲁义善,汪志文,郑琦,蔡佳,简纪常
摘要(Abstract)：
【目的】补体3(Complement3,C3)在罗非鱼的抗病感染中有重要作用,研究C3基因判断原核表达及表达条件。【方法】根据NCBI中已公布罗非鱼补体C3的基因序列,选取C3基因片段,设计一对带有酶切位点的特异性引物,经连接、转化等后,使用限制性核酸内切酶EcoRI和XhoI对克隆成功的C3基因片段以及原核表达载体pGEX-4T进行双酶切,构建重组质粒pGEX-4T-C3,导入大肠杆菌进行原核表达,并优化表达条件,并对纯化目的蛋白进行Western Blot鉴定。【结果】C3基因片段的重组质粒pGEX-4T-C3构建成功。罗非鱼C3基因片段编码区长度为1 341 bp。获得的目的蛋白分子质量为75 ku,与预期结果相符。最佳诱导时间、浓度以及温度分别为4 h、0.2 mmol/L以及37℃;目的蛋白主要以包涵体的形似存在。重组蛋白可以与GST-Tag单克隆抗体特异性结合,说明成功获得罗非鱼C3片段的重组蛋白。
关键词(KeyWords)： 尼罗罗非鱼;补体3;原核表达;条件优化
基金项目(Foundation): 国家自然科学基金(31572651)
作者(Author): 牛金中,黄瑜,汤菊芬,鲁义善,汪志文,郑琦,蔡佳,简纪常
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