| 摘要: |
| 【目的】对加州鲈弹状病毒弱毒三水株（MSRV-SS-7）及其母源株（MSRV-SS）进行全基因组克隆和测序分析。【方法】采用分段扩增方法对MSRV-SS-7及MSRV-SS基因组核心序列进行PCR扩增，第1轮根据鳜鱼弹状病毒（SCRV）基因序列（NC_008514.1）设计10对引物进行扩增并测序；在第1轮扩增产物测序、拼接基础上,设计8对引物进行第2轮扩增，拼接后获得基因组核心序列。同时用cDNA末端快速扩增法（RACE）获得3′和5′末端序列。采用Vector NTI 8.0对基因组核心序列和末端序列进行拼接，获得基因组全序列，使用Clone Manager 8.0对MSRV-SS-7和MSRV-SS进行序列比对分析；同时使用DNAMAN将基因组G基因序列转换为氨基酸序列，并通过MEGA 5.0与其他鱼类弹状病毒的G蛋白氨基酸序列进行进化树分析。【结果】MSRV-SS-7及其母源株MSRV-SS基因组全长均为11 548 bp，包含N、P、M、G、L等5个结构基因，基因组结构为3′Leader N-P-M-G-L-Trailer 5′；MSRV-SS-7与MSRV-SS全基因组中共有10处核苷酸不同。G蛋白进化分析表明，MSRV-SS-7及母源株MSRV-SS与SCRV亲缘关系最近，且与鲈鱼弹状病毒属（Perhabdovirus）聚为一支。【结论】克隆分析了MSRV-SS-7的全基因组序列，可以用于后续活疫苗的开发。 |
| 关键词: 鱼类弹状病毒 弱毒株 加州鲈 全基因组序列 G蛋白 |
| DOI： |
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| 基金项目:广东省促进经济发展专项资金（海洋经济发展用途）项目(GDME-20181007)；广东省渔港建设和渔业产业发展专项(粤财农〔2017〕17号)；广州市珠江科技新星项目（201710010087） |
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| Genome sequencing and analysis of Sanshui strain of Micropterus salmoides rhabdovirus |
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LIU Huifang,ZHANG Lijuan,LI Ningqiu,et al
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| Abstract: |
| 【Objective】An avirulent Sanshui strain of micropterus salmoides rhabdovirus (MSRV-SS-7) and its parent strain MSRV-SS were selected for genome sequence determination and analysis.【Method】The core sequences of MSRV-SS-7 and MSRV-SS genome were sequenced by fractional PCR amplification.The first round sequences were amplified with 10 pairs of primers according to the siniperca chuatsi rhabdovirus (SCRV,NC_008514.1).Then,8 pairs of primers were designed for the second round amplification based on the sequence determined in the first round,and the core sequences were obtained after splicing.The RACE end amplification method was simultaneously used to obtain the 3′ and 5′ end sequences.The genome core sequence and the end sequence were spliced using Vector NTI 8.0 to obtain the complete genome sequence,and the sequence comparative analysis was performed with MSRV-SS-7 and MSRV-SS using Clone Manager 8.0.The genomic G gene sequence was converted to amino acid sequence using DNAMAN,and phylogenetic tree analysis was performed with the G protein amino acid sequence of other fish rhabdoviruses using MEGA 5.0.【Result】Both MSRV-SS-7 and MSRV-SS genomes were 11 548 nucleotides (bp) in length.The genome encoded 5 structural genes of N,P,M,G and L.The genomic structure was 3′Leader N-P-M-G-L-Trailer 5′.The alignment analysis between MSRV-SS-7 and MSRV-SS revealed that there were 10 different nucleotides in the whole genome.The G protein phylogenetic tree analysis indicated that MSRV-SS-7 and MSRV-SS were most closely related to SCRV and classified into the genus Perhabdovirus.【Conclusion】The complete genome sequence of MSRV-SS-7 by clonal analysis can lay the foundation for the subsequent construction of the reverse genetic system for SCRV vaccine. |
| Key words: fish rhabdovirus avirulent virus Micropterus salmoides genome sequence G protein |
