Chen Jia-Jie, Xia Li-Xin, Liu Zhi-Gang, Liu Wen, Ji Kun-Mei
Allergy and Immunology Institute, Shenzhen University, Shenzhen 518060, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2008 Oct 30;26(5):356-60.
To clone the gene of arginine kinase (AK) from Periplaneta americana, produce its recombinant protein and investigate its allergenicity.
The cDNA of AK was cloned using specific primers from the total RNA of P. americana. The cloned gene was inserted into pMD18-T vector and digested by BamHI and HindIII. 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 (Ni2+) chelating affinity chromatography. Its allergenicity was examined by both Western blotting and enzyme-linked immunosorbent assay (ELISA).
The cloned cDNA ORF sequence (Accession no. EU429466) contained 1068 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 45000 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.
The recombinant cockroach arginine kinase has been obtained with proper allergenicity.
克隆美洲大蠊精氨酸激酶（AK）基因，制备其重组蛋白并研究其致敏性。
用特异性引物从美洲大蠊总RNA中克隆AK的cDNA。将克隆的基因插入pMD18-T载体，用BamHI和HindIII酶切。对cDNA进行测序并亚克隆到pET-28a表达载体中。通过IPTG诱导在大肠杆菌BL21（DE3）中表达克隆的AK cDNA基因。用金属（Ni2+）螯合亲和层析法纯化重组AK（rAK）。通过蛋白质印迹法和酶联免疫吸附测定（ELISA）检测其致敏性。
克隆的cDNA开放阅读框序列（登录号EU429466）含1068 bp，编码365个氨基酸。其核苷酸序列与已发表序列（登录号AY563004）的同源性在核苷酸水平为99.9%。在IPTG诱导下，变应原rAK在大肠杆菌BL21（DE3）中高表达为可溶性蛋白，主要分子量约为45000，并用6-His标签纯化系统纯化。在非变性和变性条件下，通过蛋白质印迹法和ELISA鉴定重组变应原与蟑螂过敏患者血清中IgE抗体的亲和力。
已获得具有适当致敏性的重组蟑螂精氨酸激酶。
