猪γ干扰素基因的合成、表达及纯化

目的通过人工合成猪γ干扰素(poIFN-γ)基因的编码序列,提高目的蛋白的表达。方法根据poIFN-γ的氨基酸序列,选择大肠杆菌所偏爱的密码子,设计并合成了24个寡核苷酸片段。通过重叠区扩增法,利用PCR合成poIFN-的成熟蛋白编码基因,克隆入pGEM-7Zf(+)载体,转化大肠杆菌TG1。经测序证实后,构建原核表达载体pBV222/poIFN-γ并转化大肠杆菌DH5α,经诱导表达后,进行SDS-PAGE分析及纯化。结果酶切及测序结果表明表达载体构建成功,工程菌诱导表达后的电泳图谱在相对分子质量约16500的位置出现明显的目的蛋白条带,表达产物主要以包涵体形式存在,约占菌体总蛋白的55%,占包涵体中总蛋白的80%,经Ni2+-NTA亲和层析纯化,纯度达90%以上。结论已获得了纯度较高的目的蛋白,为下一步对猪γ干扰素进行复性及活性研究奠定了基础。

Gene Synthesis,Expression and Purification of Porcine IFN-γ

Objective To synthesize the gene encoding porcine IFN-γ(poIFN-γ) and improved the expression of target protein.Methods Design and synthesize 24 oligonucleotide fragments with the selected E.coli-preferred codon according to the amino acid sequence of poIFN-γ,then synthesize the gene fragment encoding mature poIFN-γ protein by overlap extension PCR.Clone the synthetic gene fragment into vector pGEM-7Zf(+) and transform to E.coli TG1.Identify the cloned gene fragment by sequencing and insert into plasmid pBV222.Transform the constructed recombinant plasmid pBV222/poIFN-γ to E.coli DH5α,identify the expressed product by SDS-PAGE and purify by Ni2+ affinity chromatography.Results Restriction analysis and sequencing proved that the prokaryotic expression vector pBV222/poIFN-γ was successfully constructed.The expressed product in a form of inclusion body showed a relative molecular mass of about 16 500,contained about 55% of total somatic protein and 80% of total inclusion body protein and reached a purity of more than 90% after purification.Conclusion Highly purified poIFN-γ was obtained,which laid a foundation of refolding and further study on activity of the protein.