摘要
目的克隆并表达牛奶中主要过敏原β-乳球蛋白(β-LG)的一基因片段,并检测其抗原性。方法利用RT-PCR技术克隆β-LG蛋白基因片段,测序后将目的片段克隆入原核表达质粒pET载体,转化至大肠杆菌origami,经IPTG诱导表达,获得重组β-LG片段蛋白。用Ni2+亲和层析柱纯化,用Western blot和ELISA检测重组蛋白与牛奶过敏患者血清IgE的结合活性。结果克隆获得β-LG片段蛋白,其开放阅读框基因为264 bp(含终止密码子),编码87个氨基酸。获得的重组β-LG片段蛋白既以包涵体形式存在又以可溶性形式存在,用可溶性的蛋白进行纯化,经Western blot和ELISA检测具有较好的抗原性。结论成功地克隆和表达了β-LG的一基因片段,为后续牛奶的免疫原性研究提供参考,也为研制牛奶主要过敏原的单克隆抗体和制备其主要过敏原的检测试剂奠定了基础。
Objective To clone,express,purify a fragment of β-lactoglobulin,and to identify its allergenicity.Methods RT-PCR method was applied to clone the cDNA of a fragment of β-lactoglobulin,and then the fragment cDNA was sequenced and sub-cloned into pET expression vector.The cloned gene was expressed in E.coli origami induced by IPTG.The recombinant protein was purified by metal（Ni2 ＋） chelating affinity chromatography.The allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay（ELISA）.Results The recombinant fragment of β-lactoglobulin gene was cloned with a 264 bp open reading frame coding for 87 amino acids.The fragment of β-lactoglobulin was expressed both as inclusion bodies and soluble protein.The soluble protein was purified,and the immunogenicity of the protein identified by ELISA was good.Conclusion The clone and expression of the fragment of β-lactoglobulin was successful;which would provide references for the following studies on testing the immunogenicity of milk and lay great foundations for developing monoclonal antibodies and detection kits for the major allergens of milk.
出处
《中国食品卫生杂志》
北大核心
2011年第2期109-113,共5页
Chinese Journal of Food Hygiene
基金
深港创新圈计划项目(200701)
深圳大学团队基金(200904)
