[德国小蠊精氨酸激酶的克隆、表达、纯化及其免疫活性]
[Cloning, expression and purification of arginine kinase from Blattella germanica and its immune activity].
作者信息
Yan Hao, Xia Li-Xin, Chen Jia-Jie, Liu Jiao, Deng Zhi-Qiong, Yi Hai-Tao, Liu Xiao-Ping
机构信息
Allergy and Immunology Institute, Medical School, Shenzhen University, Shenzhen 518060, China.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2011 Jun;29(3):191-4.
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 Western blotting.
RESULTS
The cloned cDNA ORF sequence (GenBank accession No. FJ514482) contained 1071bp and encoded 356 amino acids. Its sequence homology with the published one (GenBank accession No. EU429466) was 97.2% at nucleotide level. The recombinant containing recombinant plasmid pET-28a-AK expressed a soluble protein of AK (Mr 45 000) after being induced with IPTG. The recombinant AK protein was recognized by sera of allergic patients, indicating that the recombinant AK protein has an adequate response activity.
CONCLUSION
The AK gene of Blattella germanica has been cloned and the recombinant AK protein has been confirmed with immune activity.
目的
克隆并表达德国小蠊精氨酸激酶（AK）基因，分析其免疫活性。
方法
用特异性引物从德国小蠊总RNA中克隆AK的cDNA。将AK的开放阅读框（ORF）克隆到pET-28A载体中，并用IPTG诱导在大肠杆菌BL21(DE3)中表达。重组蛋白通过Ni2+螯合亲和层析法纯化。重组蛋白经SDS-PAGE检测，其免疫活性通过Western印迹分析。
结果
克隆的cDNA ORF序列（GenBank登录号FJ514482）含1071bp，编码356个氨基酸。其核苷酸序列与已发表序列（GenBank登录号EU429466）的同源性为97.2%。含重组质粒pET-28a-AK的重组体经IPTG诱导后表达出可溶性的AK蛋白（Mr 45 000）。重组AK蛋白能被过敏患者血清识别，表明重组AK蛋白具有足够的反应活性。
结论
已克隆德国小蠊的AK基因，且重组AK蛋白已被证实具有免疫活性。
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