摘要
为了解锦鲤疱疹病毒中国吉林株(KHV-CJ)ORF56基因的结构和序列特征,试验采用锦鲤尾鳍原代细胞增殖培养KHV-CJ,提取DNA,以其为模板经PCR扩增获得ORF56基因,将该基因克隆到PVAX表达载体上,构建重组质粒PVAX-ORF56,并体外表达重组质粒,对KHV-CJ ORF56基因序列与GenBank上已知的对应基因序列进行比对,应用生物信息学方法初步分析KHV-CJ ORF56基因的结构和功能。结果表明:成功克隆出KHV-CJ ORF56基因;双酶切鉴定显示,目的基因与PVAX表达载体连接;体外表达重组质粒可见绿色荧光;与GenBank上已知序列比对显示,同源性达到99.9%;该基因序列信号肽切割部位最可能位于第26位的组氨酸和谷氨酸之间,没有跨膜区,最大疏水指数为2.174,最小疏水指数为-0.334,其抗原表位主要集中在第1~27,54~143,156~223,254~295位氨基酸。说明KHV-CJ ORF56基因是较好的抗原候选基因。
In order to understand the structure and sequence characteristics of the Chinese Jilin strain(KHV-CJ) ORF56 gene of Koi Herpes Virus, KHV-CJ was cultured by proliferation of primitive cells of Koi caudal Fin, and DNA was extracted. The ORF56 gene was obtained by PCR amplification.The gene was cloned into the PVAX expression vector to construct the recombinant plasmid PVAX-ORF56, and the recombinant plasmid was expressed in vitro to compare the KHV-CJ ORF56 gene sequence with the corresponding gene sequence known on GenBank. The structure and function of KHV-CJ ORF56 gene were analyzed by bioinformatics method. The results showed that KHV-CJ ORF56 gene was successfully cloned. Double enzyme identification showed that the target gene was connected with PVAX expression vector. In vitro expression of recombinant plasmid can be seen as green fluorescence. Compared with the known sequence on GenBank, the homology reached 99.9%. The gene sequence signal peptide cutting site was most likely located between histidine and glutamic acid at the 26 th position, there was no transmembrane area, the maximum hydrophobic index was 2.174, the minimum hydrophobic index was-0.334, and its antigen site was mainly concentrated on 1-27, 54-143, 156-223, 254-295 amino acids. It shows that the KHV-CJ ORF56 gene is a good antigen candidate gene.
引文
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