蛋白G IgG Fc段结合域的克隆表达及功能研究

Cloning,Expression and Function of Protein G IgG Fc Binding Domain

旨在研究蛋白G IgG Fc段结合域(PGFB)的克隆、表达及其抗体结合功能,用于抗体的纯化。根据PGFB的氨基酸序列,选择大肠杆菌偏爱的密码子,设计并合成了4个寡核苷酸片段。通过重叠延伸PCR方法合成了PGFB DNA片段,测序鉴定后克隆至原核表达系统pET-28a-c(+)上,转化大肠杆菌,获得表达菌株;IPTG诱导表达PGFB,经Ni+-NTA琼脂糖凝胶层析纯化后偶联到琼脂糖凝胶6B上,用其纯化多克隆抗体。结果显示,PGFB在大肠杆菌BL21(DE3)中获得高效表达,纯化后纯度达到90%以上,相对分子量为12.25 kD,与预期值相符。此外,偶联产物纯化多克隆抗体达到了良好的效果,每毫升基质可结合20 mg抗体。本研究克隆构建并高效表达了具有较好抗体亲和能力的PGFB,为多克隆抗体的快速纯化提供了方便。

It was to clone and express protein G IgG Fc binding domain（ PGFB ）for purifying antibody. Four oligonucleotide fragments with the selected E. coli-preferred codon were designed and synthesized according to the amino acid sequence of PGFB. Then the gene of PGFB was synthesized by overlap extension PCR, and cloned into pET-28a-c（ ＋ ） plasmid. The recombinant protein was purified by a single step affinity chromatography with Ni^＋ -NTA, then it was coupled with Sepharose 6B to purify polyclonal antibody. Results showed PGFB was expressed highly in E. coli BL21 （ DE3 ）, the purity of which reached 90% with expected molecular weight of 12.25 kD. PGFB coupled with Sepharose 6B which can purify polyclonal antibody. The binding capacity reached 20 mg/mL. Therefore, highly expressed PGFB would provide convenience for the rapid purification of polyclonal antibody.