犬传染性肝炎病毒（ICHV）主要的中和抗原表位位于六邻体蛋白Loop1、Loop2上。本次研究参考Genebank发表的基因序列设计引物，提取ICHV基因组DNA，，分别PCR扩增六邻体蛋白（Hexon）的Loop1、Loop2基因片段，用T4酶连接在一起，克隆入原核表达载体pET28a中，测序显示本室保存病毒分离株Loop1与CLL株、RI261株和Toronto A26/61株核苷酸序列同源性分别为100%、100%和83.8%；Loop2与CLL株、RI261株和Toronto A26/61株核苷酸序列同源性分别为88.1%、88.1%和99.3%，推导的氨基酸序列同源性分别为93.6%、93.6%和98.6%。转化BL21工程菌，实现了重组Loop蛋白在大肠杆菌中的高效表达，其表达的重组蛋白以包涵体形式存在，分子量约为36kDa，并且利用镍柱纯化重组蛋白，纯度达95%以上。本实验为建立新的犬传染性肝炎病毒基因工程产品奠定了良好的基础。
Variations in the amino acid sequence of Infectious canine hepatitis virus (ICHV) structural proteins are the molecular basis for the antigen diversity of the virus.Majority of antigenic sites for the virus neutraliation are present on Loop1 and Loop2 of hexon.ICHV (the isolated strain) DNA was isolated and purified from the cultured MDCK cells.The Loop1 and Loop2 fragments were amplified by polymerase chain reaction(PCR) method,and then was connected by ligase T4.The target fragment was then connected with vector pET28a.The nucleotide sequence ecoding Loop1 and Loop2 was determined.The nucleotide sequence identity of Loop1 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 100%, 100% and 83.8%, and the nucleotide sequence identity of Loop2 region between the isolated strain and CLL, RI261 and Toronto A26/61 strains is 88.1% , 88.1% and 99.3%, and amino acid identity is 93.6 , 93.6% and 98.6%.The recombinent Loop protein was expressed in E. coli and was approximately 36kDa in size,and then was purified.
郑龙,王俊霞,李丽敏,张霞,张焕铃,尤红煜. 犬传染性肝炎病毒Hexon蛋白Loop1、Loop2基因的克隆与表达[J]. 中国生物工程杂志, 2007, 27(4): 29-33.
. Clone and expression of Loop1 and Loop2 gene of hexon of infectious canine hepatitis virus. China Biotechnology, 2007, 27(4): 29-33.
https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2007/V27/I4/29
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