摘要
目的:克隆并表达牛奶中主要过敏原αs1-酪蛋白的全长及N端、C端两个片段区基因,用于筛选与制备牛奶主要过敏原的单克隆抗体。方法:利用RT-PCR技术克隆αs1-酪蛋白基因,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(E.coli)BL21(DE3)。经IPTG诱导表达,获得重组αs1-酪蛋白。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αs1-酪蛋白全长的开放阅读框基因为645bp(含终止密码子),编码214个氨基酸。N、C端片段区基因(含终止密码子)分别为285、294bp,编码94、97个氨基酸。三种重组蛋白均能以可溶性表达形式纯化,经Western blot和ELISA检测都具有较好的免疫原性,而αs1-酪蛋白全长的免疫原性更强。结论:成功地克隆和表达了αs1-酪蛋白全长及N、C端两个片段区基因,为研制牛奶主要过敏原的单克隆抗体,制备牛奶主要过敏原的检测试剂奠定了基础。
Objective：To clone,express and purify the full-length and fragments of the αs1-casein.Methods：RT-PCR was applied to clone cDNA of αs1-casein,and then the full-length and fragments cDNA were sequenced and subcloned into pET expression vector.The cloned genes were expressed in E.coli BL21 （DE3） by IPTG induction.The recombinant αs1-casein products were purified by metal （Ni2＋） chelating affinity chromatography.Their allergenicity was examined by both Western blot and ELISA.Results：The recombinant αs1-caseins was cloned with a 645 bp open reading frame coding for 214 amino acids.The fragment of N and C terminal were 285 bp and 294 bp coding for 94 and 97 amino acids respectively.They were all expressed as soluble protein and have good immunogenicity,but the full-length was better.Conclusion：Three recombinant αs1-caseins products have been successfully expressed.The study will make great foundations for developing the monoclonal antibodies and the detection kit of the major allergens of milk.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2010年第7期601-605,共5页
Chinese Journal of Immunology
基金
深港创新圈计划项目(200701)
深圳大学团队基金(200904)
