中文摘要：

目的克隆美洲大蠊（Periplaneta americana）精氨酸激酶（arginine kinase,AK）基因并表达、纯化具有变应原活性的重组AK。方法提取美洲大蠊总RNA,设计特异引物,通过RT-PCR克隆美洲大蠊AK基因目的片段,测序后将该片段克隆入原核表达载体pET-28a,转化至大肠埃希菌（E.coli）BL21（DE3）,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,获重组AK。用镍离子（Ni2＋）亲和层析柱纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组AK表达情况。用蛋白质印迹分析（Western blotting）（变性条件）和ELISA（非变性条件）检测重组AK变应原与过敏患者血清IgE结合活性,并以健康人血清为对照。结果测序结果表明,AK基因含有1068bp的开放阅读框,编码356个氨基酸（登录号为EU429466）。与Gen Bank公布的序列（登录号为AY563004）比较同源性达99.9%。SDS-PAGE分析表明,该变应原基因在E.coli中主要以可溶形式高水平表达,相对分子质量约为Mr45000。重组变应原AK在变性与非变性条件下,与过敏患者血清IgE均具有良好的结合活性,与健康人对照组之间的差异具有统计学意义（P〈0.05）。结论获得了具有变应原活性的重组美洲大蠊精氨酸激酶。

英文摘要：

Objective To clone the gene of arginine kinase （AK） from Periplaneta americana, produce its recombinant protein and investigate its allergenicity. Method The cDNA of AK was cloned using specific primers from the total RNA ofP. americana The cloned gene was inserted into pMD18-Tvector and digested by BamHl and HindllI. The cDNA was sequenced and subcloned into pET-28a expression vector. The cloned AK cDNA gene was expressed in Escherichia coli BL21 （DE3） by IPTG induction. The recombinant AK （rAK） was purified by metal （Ni^2＋） chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay （ELISA）. Result The cloned cDNA ORF sequence （Accession no. EU429466） contained 1 068 bp and encoded 365 amino acids. Its sequence homology with the published one （Accession no. AY563004） was 99.9% at nucleotide level. The allergen rAK was highly expressed in E. coli BL21 （DE3） as a soluble protein mainly with the molecular weight of about Mr 45 000 under induction of IPTG and purified by 6-His-tag purification system. Both in the non-denaturalization and denaturalization conditions, the recombinant allergen was identified as its affinity to IgE antibodies from the cockroach-allergic patient sera by Western blotting and ELISA. Conclusion The recombinant cockroach arginine kinase has been obtained with proper allergenicity.