鸡蛋主要过敏原卵类粘蛋白基因的克隆及原核载体构建与表达  

Gene cloning,prokaryotic vector constructing for the major allergen ovomucoid in Hen's egg white and its recombinant expression

作　　者：陈大玮[1] 邬玉兰[1] 刘志刚[1] 吴序栎[1] 孔小丽[1] 

机构地区：[1]深圳大学医学院过敏反应与免疫学研究所,深圳518060

出　　处：《中国免疫学杂志》2009年第9期828-831,共4页Chinese Journal of Immunology

基　　金：国家自然科学基金(No.30871752);深圳市深港创新圈项目;深圳大学校科研基金(No.200712;200807);国家质检总局项目

摘　　要：目的:本研究利用PET表达载体克隆,表达鸡蛋主要过敏原卵类粘蛋白基因,为鸡蛋过敏疾病的特异性诊断和治疗以及进一步的实验研究奠定了一定的基础。方法:提取鸡输卵管组织总RNA,利用RT-PCR技术扩增卵类粘蛋白基因(ovomucoid,OVM),并与已知序列进行同源性比较,将该片段连接入原核表达载体pET-28a,IPTG诱导表达目的蛋白。结果:成功克隆鸡蛋主要过敏原卵类粘蛋白(ovomucoid,OVM)的全长基因,该基因的开放阅读框长度为633bp(包括终止密码子),编码210个氨基酸,与GenBank提供的OVM基因序列同源性达99%。该序列编码的蛋白为小分子量蛋白,相对分子质量约为21kD,与理论值均相符。IPTG诱导表达后,SDS-PAGE分析表明目的蛋白在宿主大肠杆菌BL21(DE3)中高效表达。结论:本研究采用体外重组的方法克隆出卵类粘蛋白基因,并实现在大肠杆菌中大量表达,这为后续鸡蛋食品基础性研究奠定基础。Objective： To clone and express the gene of ovomucoid, which is the main allevgen in egg white. Methods： Using total RNA of chicken oviduct as template, the gene of ovomucoid was amplified by RT-PCR. The homology was analyzed by comparision with the sequence in GeneBank. Subsequently, the PCR product of the ovomucoid gene was cloned into prokaryotic expressing vector pET-28a and was expressed by the challenge of IPTG. Results： The whole gene of ovomucoid, one of the main allergens in egg white, was successfully cloned. The cloned ORF sequence contains 633 bp, including stop codon, encods for 210 amino acids. Sequence analysis shows that the ovomucoid gene displays 99% nucleotide identities with the published sequences. The molecule weight of ovomucoid protein obtained was 21 kD. By the challenge of IPTG, SDS-PAGE analysis showed that the ovomucoid gene was overexpressed in E. coli BL21 （DE3）. Conclusion： The gene of ovomucoid is cloned and overexpressed in E. coli BL21（DE3）.This study will be the basis for the further research.

关 键 词：鸡蛋 卵类粘蛋白 基因克隆 原核载体 表达 

分 类 号：R392.8[医药卫生—免疫学]