大豆主要过敏原Gly m Bd 30K的抗原表位区基因的克隆表达、纯化及免疫原性鉴定

Cloning and Expression of the Antigenic Epitope of Gly m Bd 30K Protein from Soybean and Purification and Identification of Expressed Product

根据文献并结合生物信息学方法选取大豆主要过敏原Gly m Bd 30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(DE3)plysS,经IPTG诱导后进行SDS-PAGE分析;同时用Ni2+离子亲和层析柱纯化表达的sGly和dGly抗原表位蛋白,用western-blotting和ELISA检测重组蛋白的免疫原性。结果表明:表达的单体sGly蛋白以可溶性表达为主,而其二聚体dGly蛋白以包涵体形式存在,纯化的sGly和dGly蛋白都具有较好的免疫原性,重组蛋白sGly的抗原性更佳。成功构建的大豆主要过敏原Gly m Bd 30k蛋白的抗原表位区单体sGly蛋白及其二聚体dGly蛋白的工程菌,为研制大豆主要过敏原的单克隆抗体以制备用于大豆主要过敏原检测的试剂奠定了基础。

Through reviewing the literatures combined with bioinformatics predication, the antigenic epitope of Gly m Bd 30k allergen was selected. And the geue was amplified by PCR. The pET expression vector for the monomer protein （pET-sGly） and the dimer （pET-dGly） were constructed. The recombinant proteins were expressed in E. coli BL21 （DE3） plysS by IPTG, analyzed by SDS-PAGE, and then purified by metal （ Ni^2＋ ） chelating affinity chromatography. The immunogenicity was tested by western-blotting and ELISA. Results shows that the monomer protein sGly was mainly expressed as soluble protein, while the dimer protein dGly as inclusion bodies. Purified protein sGly and dGly both have good immunogenicity, and sGly is better. The prokaryotic expression vectors for the monomer protein and dimer of the antigenic epitope of Gly m Bd 30k protein of soybean will make great foundations for developing the monoclonal antibodies and the detection kit of the major allergens of soybean.