尼罗罗非鱼补体3基因片段的原核表达及条件优化

摘要

[Objective]Because complement 3 (C3) plays an important role in disease-resistant infection of tilapia, this study aim to optimize the production of recombinant C3 by a prokaryotic expression system for future disease control studies.[Method]According to the functional domain sequence of the cDNA segment of tilapia complement 3 gene in NCBI, we selected a pair of specific primers with cleavage site by C3 gene segment.After ligation and transformation,the recombinant C3 gene segment and the prokaryotic expression vector pGEX-4T were digested with restriction endonucleases EcoRI and Xho I,the recombinant plasmid pGEX-4T-C3 of C3 gene segment was constructed and introduced into Escherichia coli to detect the target protein. Then the optimum gene expression conditions were optimized, and the target protein was identified by Western Blot.[Result]The recombinant plasmid pGEX-4T-C3 of C3 gene segment was successfully constructed.The length of coding region of tilapia C3 gene segment was 1 341 bp. The molecular weight of target protein was about 75 ku, which was consistent with the expected results. The optimal time, concentration and temperature were 4 h, 0.2 mmol/L and 37 ℃, respectively. The target protein was mainly in the form of inclusion bodies. The recombinant protein can specifically bind to GST-Tag monoclonal antibody.%[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1341 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.