| 德国小蠊精氨酸激酶基因的克隆、表达及免疫活性测定 |
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| 引用本文： | Yan H,Xia LX,Chen JJ,Liu J,Deng ZQ,Yi HT,Liu XP. 德国小蠊精氨酸激酶基因的克隆、表达及免疫活性测定[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(3): 191-194 |
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| 作者姓名： | Yan H Xia LX Chen JJ Liu J Deng ZQ Yi HT Liu XP |
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| 作者单位： | 深圳大学医学院过敏反应与免疫学研究所;中南大学湘雅二医院; |
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| 基金项目： | 国家自然科学基金(No.30271226)~~ |
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| 摘 要： | 目的克隆德国小蠊精氨酸激酶(arginine kinase,AK)全长基因,表达、纯化重组AK蛋白,并研究蛋白的免疫反应性。方法提取德国小蠊总RNA,反转录为cDNA,设计特异性引物,PCR克隆德国小蠊AK片段,将测序正确的目的片段克隆至原核表达载体pET-28a中,在大肠埃希菌(E.coli)BL21(DE3)中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过镍离子亲和层析纯化目的蛋白。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测目的蛋白表达和纯化结果,用蛋白质印迹(Western blotting)分析重组AK蛋白的免疫反应性。结果德国小蠊AK基因开放阅读框全长为1071bp,编码356个氨基酸,GenBank登录号为FJ514482。与GenBank中已登录的德国小蠊序列(登录号为EU429466)比对,同源性达97.2%。重组质粒pET-28a-AK在E.coli BL21(DE3)中获得高效表达,重组蛋白相对分子质量(Mr)约为45 000,主要以可溶性形式表达,经亲和层析获得目的蛋白。Western blotting分析结果表明,重组AK蛋白可被过敏性患者血清识别,免疫反应...
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| 关 键 词： | 德国小蠊 精氨酸激酶 过敏原 克隆 |
Cloning, expression and purification of arginine kinase from Blattella germanica and its immune activity |
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| Yan Hao,Xia Li-Xin,Chen Jia-Jie,Liu Jiao,Deng Zhi-Qiong,Yi Hai-Tao,Liu Xiao-Ping. Cloning, expression and purification of arginine kinase from Blattella germanica and its immune activity[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2011, 29(3): 191-194 |
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| Authors: | Yan Hao Xia Li-Xin Chen Jia-Jie Liu Jiao Deng Zhi-Qiong Yi Hai-Tao Liu Xiao-Ping |
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| Affiliation: | YAN Hao 1,XIA Li-xin 1,CHEN Jia-jie 1,LIU Jiao 1,DENG Zhi-qiong 1,YI Hai-tao 1,LIU Xiao-ping 2 *(1 Allergy and Immunology Institute,Medical School,Shenzhen University,Shenzhen 518060,China,2 The Second Xiangya Hospital,Central South University,Changsha 410011,China) |
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| Abstract: | Objective To clone and express the arginine kinase(AK) gene of Blattella germanica and analyze its immune activity.Methods The cDNA of AK was cloned using specific primers from the total RNA of Blattella germanica.The open reading frame(ORF) of AK was cloned into pET-28A vector,and expressed in Escherichia coli BL21(DE3) with IPTG induction.The recombinant protein was purified by Ni2+ chelating affinity chromatography.The recombinant protein was detected by SDS-PAGE,and its immune activity was analyzed by W... |
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| Keywords: | Blattella germanica Arginine kinase Allergen Clone |
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