作　　者： ; ; ; ; ;

机构地区： 深圳大学生命科学学院

出　　处： 《应用昆虫学报》 2013年第1期133-138,共6页

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。 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.

关 键 词： 东亚飞蝗 精氨酸激酶 过敏原 克隆表达 纯化

领　　域： [生物学]