太平洋牡蛎过敏原精氨酸激酶的分子克隆、表达纯化和免疫原性鉴定  

Cloning,expression,purification and immunogenicity identification of arginine kinase,the allergen from Crassostrea gigas

作　　者：赵晓涵 刘文颖[1] 程青丽 谷瑞增[1] 鲁军[1] 李国明[1] ZHAO Xiao-han;LIU Wen-ying;CHENG Qing-li;GU Rui-zeng;LU Jun;LI Guo-ming(China National Research Institute of Food&Fermentation Industry,Beijing Engineering Research Center of Protein and Functional Peptides,Beijing 100015)

机构地区：[1]中国食品发酵工业研究院,北京市蛋白功能肽工程技术研究中心,北京100015

出　　处：《中国食品添加剂》2021年第2期100-106,共7页China Food Additives

基　　金：国家自然科学基金项目(31671963);中国食品发酵工业研究院强院工程培育专项(院强院20-功能肽-505)。

摘　　要：克隆太平洋牡蛎精氨酸激酶(CG-AK)基因,导入大肠杆菌(BL21(DE3))中,然后筛选重组菌株。通过SDS-PAGE对重组菌株中太平洋牡蛎精氨酸激酶(CG-AK)的诱导表达条件进行探究,经Ni2+亲和层析柱纯化,通过SDS-PAGE、质谱(MALDI-TOF-MS)、圆二色(CD)光谱等方法从相对分子量、一级结构以及二级结构综合鉴定了重组CG-AK的准确性,同时使用SDS-PAGE和Western blot检测重组CG-AK的表达,并利用酶联免疫吸附(ELISA)对其免疫原性进行鉴定。成功克隆表达并鉴定了CG-AK,其最佳诱导表达条件为18℃诱导14h,具有良好的免疫原性。为国内过敏原的标准化生产提供理论支持,也为后续牡蛎过敏疾病的精准诊断、治疗及脱敏方法奠定了良好的分子生物学基础。The Pacific oyster arginine kinase(CG-AK)gene was cloned into E.coli(BL21(DE3)),and the recombinant strain was screened.The inducible expression conditions of the recombinant CG-AK were investigated by SDS-PAGE.The recombinant CG-AK was purified by Ni2+affinity chromatography column,which was comprehensively identified from the relative molecular weight,primary structure and secondary structure by SDSPAGE,mass spectrometry(MALDI-TOF-MS),circular dichroism(CD)spectroscopy.At the same time,SDS-PAGE and Western blot were used to detect the expression of recombinant CG-AK,and enzyme-linked Immunoadsorption(ELISA)was used to identify its immunogenicity.The optimal induction expression condition of CG-AK was 18℃for 14h,which was successfully cloned,expressed and identified and has good immunogenicity.It provides theoretical support for the standardized production of domestic allergens,and also lays a good molecular biology foundation for the follow-up accurate diagnosis,treatment and desensitization methods of oyster allergies.

关 键 词：太平洋牡蛎 精氨酸激酶 分子克隆 表达条件 免疫原性 过敏原 

分 类 号：R392.11[医药卫生—免疫学] TS201.6[医药卫生—基础医学]