SCMRP基因原核表达及多克隆抗体制备

SCMRP基因是根据大豆密码子偏向性,采用生物信息学方法对玉米胚乳蛋白基因(MRZP)进行改造并合成的新基因,是适合在大豆中高效表达的高含硫氨基酸基因,可用于豆科作物的品质改良。将新合成的SCMRP基因构建到含有6×His标签序列的原核表达载体pET-32b上,转化大肠杆菌BL21(DE3)菌株中,SDS-PAGE和Western blot分析表明:经IPTG诱导,转化的大肠杆菌BL21(DE3)菌株中能正常表达出约20ku的目标蛋白质,IPTG最佳诱导表达时间是6h,融合蛋白质以可溶组分形式存在。菌体总蛋白经金属鳌合层析纯化后免疫新西兰大耳兔,制备兔抗血清,经Western blot和琼脂板双向扩散试验表明,已纯化出SCMRP-6×His融合蛋白质,成功制备出SCMRP兔多克隆抗体,抗血清效价达到1:256。上述结果表明,新合成的SCMRP基因是能够在生物体中正常表达的完整基因,制备的SCMRP兔多克隆抗体可用于转SCMRP基因植物蛋白质表达分析。研究结果将为通过转基因技术改良豆科植物含硫氨基酸品质奠定重要基础。

Protokaryotic expression of SCMRP gene and preparation of polyclonal antibody

The SCMRP gene is a kind of storage protein gene coding high-sulfur-containing amino acid, which was designed and synthesized according to the soybean coden usage. The SCMRP gene was inserted in to prokaryotic expression vector pET-32b, transformed to E.coli (DE3), and the 6xHi tag was added to the C-terminal of the SCMRP protein. The analysis of SDS-PAGE and Westam blol showed that SCMRP could be successfully expressed in E.coli and the 6xHis-SCMRP fused protein was mainly existing in the soluble composition. This fused protein was isolated and purified through metal chelate chromatography, and then was used to immunize rabbits. Polyclonal antibody was prepared, the antibody titer of which was 1:256. The results showed that the new synthesized gene SCMRP could be successfully expressed in E.cofi, and the polyclonal against SCMRP could be used to analyze the expression of the protein in transgenic plant with SCMRP, both of which provided reliable tools for the qualitative breeding that used the transgenic technology to improve the sulfur-containing amino acid.