CGSIV病毒ORF22R序列分析,克隆及大肠杆菌表达

Analysis,cloning,expression of CGSIV ORF22R in E. coli

中国大鲵虹彩病毒是近年来造成驯养大鲵大规模的发病及死亡的病原体之一．以病毒基因组为模板，分离了ORF22R DNA序列．蛋白氨基酸序列信息分析结果表明中国大鲵虹彩病毒与蛙病毒属中类两栖类蛙病毒组的成员有很高的同源性，达到95.9％～98.2％；而与蛙病毒属类GIV病毒组成员的同源性相对较低，为31.7％；与淋巴囊肿病毒属的成员的同源性最低，只有16％左右．将PCR技术分离的ORF22R DNA序列克隆至原核表达质粒pET22b．重组质粒pET22b-ORF22R转化于 Rosetta 大肠杆菌菌，经 IPTG诱导，表达重组蛋白．镍柱纯化后 SDS-PAGE检测ORF22R蛋白分子量为65 kD，CGSIV ORF22R蛋白原核表达成功．

Recently,Infectious diseases are implicated in the declines and the economic loss in cul-tured and wild Chinese giant salamander.One of pathogens has been recognized as Chinese giant sala-mander iridovirus （CGSIV）.The putative ORF22R of CGSIV was amplified and sequenced.The ORF consists of 1 818 bp,which codes for a protein of 605 aa with a predicted molecular mass of 65.185 kD. Comparative studies of the identity of the amino acid sequence of ORF22R were carried out with different iridovirus species.Alignment of the identity of the amino acid sequence of this ORF of CGSIV and amphib-ian-like ranaviruses（ALRV）showed 95.9%-98.2% identity,but significantly higher than that of GIV-like viruses （31.7%）and the Lymphocystis disease virus 1 and C in other genus of Ranavirus （~16%）. ORF22R was cloned into the pET22b vector,and expressed in E.coli Rosetta.The protein ORF22R was purified with nickel column chromatography after inclusion body lysis,and isolated by SDS-PAGE.