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当前位置: 首页 > 文章 > 东亚飞蝗主要过敏原精氨酸激酶基因的克隆表达、纯化及免疫原性鉴定 应用昆虫学报 2013,50 (1) 133-138
Position: Home > Articles > Cloning,expression and purification of arginine kinase from Locusta migratoria manilensis and its allergic activity Chinese Journal of Applied Entomology 2013,50 (1) 133-138

东亚飞蝗主要过敏原精氨酸激酶基因的克隆表达、纯化及免疫原性鉴定

作  者：
陈义昆;邬玉兰;李荔;连国云;刘志刚
单  位：
深圳大学医学院;深圳大学生命科学学院
关键词：
东亚飞蝗;精氨酸激酶;过敏原;克隆表达;纯化
摘  要：
本研究克隆了东亚飞蝗Locusta migratoria manilensis(Meyen)精氨酸激酶(arginine kinase,AK)基因全长,表达并纯化重组AK蛋白,研究重组蛋白的免疫反应性。东亚飞蝗AK基因开放阅读框全长为1 068 bp,编码355个氨基酸,与GenBank中已登录的东亚飞蝗AK(DQ513322)基因同源性为98%,重组质粒pET-28a-AK在E.coli中获得高效表达,重组蛋白相对分子质量(Mr)约为40 000,主要以可溶性形式表达,经亲和层析获得重组蛋白。通过免疫印迹分析结果表明,重组AK蛋白可被过敏性患者血清识别,免疫原性良好。结果表明我们成功获得东亚飞蝗精氨酸激酶全长基因并表达出重组AK蛋白,重组AK蛋白具有良好的免疫反应性。
译  名：
Cloning,expression and purification of arginine kinase from Locusta migratoria manilensis and its allergic activity
作  者：
CHEN Yi-Kun;WU Yu-Lan;LI Li1LIAN;Guo-Yun;LIU Zhi-Gang;College of Life Sciences,Shenzhen University;School of Medicine,Shenzhen University;
关键词：
Locusta migratoria manilensis;;arginine kinase;;allergen;;gene expression;;purification
摘  要：
This investigation aimed to clone the Locusta migratoria manilensis arginine kinase( AK) gene,produce its recombinant protein and investigate the protein's allergenicity. The cDNA of AK was cloned,using specific primers,from the total RNA of L. m. manilensis. The cloned gene was inserted into pMD18-T vector and digested by EcoR I and Xho I. The cDNA was sequenced and subcloned into a pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli Rosetta by IPTG induction. The recombinant AK( rAK) was purified by metal( Ni2 +) chelating affinity chromatography. Its allergenicity was examined by Western blotting. The cloned cDNA ORF sequence contained 1 068 bp and encoded 355 amino acids. Its sequence homology with the published sequence( Accession no. DQ513322) was 98% at nucleotide level. The allergen rAK was highly expressed in E. coli as a soluble protein with a molecular weight of about Mr 40 000 under induction with IPTG and purified by a 6-His-tag purification system. Under both non-denaturalization and denaturalization conditions,the recombinant allergen was identified by its affinity to IgE antibodies from the cockroach- allergic patient sera by Western blotting. It is concluded that recombinant arginine kinase with proper allergenicity was successfully obtained.

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