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目的构建表达呼吸道合胞病毒(RSV)G蛋白片段(来自130-230氨基酸)和来自链球菌蛋白G(SPG)血清白蛋白结合区(BB)的原核表达质粒,寻找适合的表达和纯化条件,获得融合表达蛋白,为进行体内外实验检测奠定基础.方法利用PCR技术,通过提取病毒cDNA和链球菌基因组DNA为模板扩增目的片段,构建融合表达质粒PET32a(BBG2Na)。转化宿主菌E. coli Bl21(DE3)进行表达,并采用亲和层析纯化目的蛋白。结果Thx-G2Na-BB在E. coli BL21(DE3)中可获得高效表达,表达量可达菌体蛋白的68.3%,并以可溶性的形式表达,纯化后的目的蛋白纯度可达90%。结论实现Thx-G2Na-BB的融合表达,此蛋白可作为抗原用于重组蛋白疫苗的筛选。
Objective To construct prokaryotic expression plasmids expressing G protein fragments (from 130-230 amino acids) of respiratory syncytial virus (RSV) and serum albumin binding domain (BB) from streptococcal protein G (SPG), and to find suitable expression and purification conditions, And obtain the fusion expression protein, which will lay the foundation for the in vitro and in vivo experiments.Methods The fusion gene expression vector PET32a (BBG2Na) was constructed by PCR amplification of viral cDNA and Streptococcus genomic DNA. The host strain was transformed into E. coli Bl21 (DE3) for expression, and the target protein was purified by affinity chromatography. Results High expression of Thx-G2Na-BB was obtained in E. coli BL21 (DE3). The expressed amount of Thx-G2Na-BB reached 68.3% of that of the bacterial protein and expressed in soluble form. The purity of the purified protein was 90%. Conclusion The fusion expression of Thx-G2Na-BB was achieved. This protein could be used as antigen in the screening of recombinant protein vaccine.