锦鲤疱疹病毒-CJ株ORF56基因的真核表达及生物信息学分析
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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.
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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[2] 张瑞雪,周井祥,王好,等. 鲤疱疹病毒3型研究进展[J]. 科学养鱼,2016, 34(6):54-56.
[3] 王好,周井祥,王秋举,等. 锦鲤疱疹病毒-CJ株ORF136基因的原核表达及免疫原性研究[J]. 中国兽药杂志,2013,47(10):6-9.
[4] 王好,王秋举,刘艳辉,等. 锦鲤疱疹病毒-CJ株ORF126基因的原核表达及免疫试验研究[J]. 中国兽医杂志,2014, 50(12):70-73.
[5] 袁海延,于慧,周井祥,等. 锦鲤疱疹病毒ORF146基因的克隆、表达及免疫原性的研究[J]. 中国兽药杂志,2015,49(6):13-16.
[6] 周井祥,李新伟,朱霞,等. 锦鲤疱疹病毒CJ株ORF25基因的克隆及生物信息学分析[J]. 中国兽药杂志,2012, 46(3):1-4.
[7] 刘建平,周井祥,王好,等. 鲤鱼Ⅲ型鲤疱疹病毒病的简述[J].科学养鱼,2014(10):56-57.
[8] ZHOU J X,WANG H,LI X W, et al. Construction of KHV-CJ ORF25 DNA vaccine and immune challenge test [J]. J Fish Dis,2014,37(4):319-325.
[9] 黄东东,于慧,李改娟,等. 感染锦鲤疱疹病毒的鲤鱼免疫相关分析[J].黑龙江畜牧兽医,2017(07下):195-198.
[10] 邢程,王好,王友超,等. 一例冰下低温爆发的锦鲤疱疹病毒的鉴定[J].水产学杂志,2014,27(1):46-49.
[11] ZHOU J, XUE J,WANG Q J,et al. Vaccination of plasmid DNA encoding ORF81 gene of CJ strains of KHV provides protection to immunized carp [J]. In Vitro Cell Dev Biol Anim,2014,50(6):489-495.