猪繁殖与呼吸综合征病毒贵州株GZ-R NSP2基因的克隆与遗传变异分析

doi:10.16431/j.cnki.1671-7236.2020.11.029

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3659-3666.doi: 10.16431/j.cnki.1671-7236.2020.11.029

猪繁殖与呼吸综合征病毒贵州株GZ-R NSP2基因的克隆与遗传变异分析

张喜懿^1,2, 温贵兰^1,2, 管国丹^1,2, 陈广^1,2, 田浪^1,2, 杨佰启^1,2

1. 贵州大学动物科学学院, 贵阳 550025;
2. 贵州省动物生物制品工程技术研究中心, 贵阳 550016

收稿日期:2020-03-24 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 温贵兰 E-mail:524340732@qq.com
作者简介:张喜懿(1995-),女,贵州铜仁人,硕士生,研究方向:微生物与免疫学,E-mail:1748891872@qq.com
基金资助:
贵州省农业科技支撑计划项目（黔科合支撑[2019]2286号）；贵州大学博士基金项目（贵大人基合字[2013]12号）；贵州省高层次创新型人才培养项目（黔财教[2017]号）；贵州省科技平台及人才团队计划项目（黔科合平台人才[2018]5253）；贵州省研究生教育创新计划项目（GZZ2017002）

Cloning and Genetic Variation Analysis of NSP2 Gene of Porcine Reproductive and Respiratory Syndrome Virus Guizhou Strain GZ-R

ZHANG Xiyi^1,2, WEN Guilan^1,2, GUAN Guodan^1,2, CHEN Guang^1,2, TIAN Lang^1,2, YANG Baiqi^1,2

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Guizhou Animal Biological Engineering Technology Research Center, Guiyang 550016, China

Received:2020-03-24 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 为了解猪繁殖与呼吸综合征病毒（PRRSV）贵州株GZ-R的遗传变异情况及其NSP2基因的特征，本试验基于NSP2基因设计了2对特异性引物对目的基因进行巢式RT-PCR、克隆和序列分析。结果显示，第二轮巢式PCR除扩增出约3 000 bp的预期条带外，还扩增出另一条约1 200 bp的条带；将两DNA片段克隆至pMD19-T载体后经测序得到两条片段，大片段长2 980 bp，命名为GZR-NSP2-L；小片段长1 131 bp，命名为，GZR-NSP2-S；两片段同源性比对发现，GZR-NSP2-S具有GZR-NSP2-L N端第1-945位核苷酸与C端第2 795-2 980位核苷酸，缺失GZR-NSP2-L第946-2 794位之间的核苷酸；GZR-NSP2-L与VR-2332株、Lelystad virus株的核苷酸同源性分别为81.2%、48.6%；编码氨基酸同源性比对结果与核苷酸同源性结果一致，揭示PRRSV GZ-R株属于美洲型毒株；氨基酸缺失位点比对发现，GZ-R NSP2在其第481位、第533-561位存在30个不连续氨基酸的缺失，缺失位点与PRRSV高致病性毒株一致。综上所述，可鉴定PRRSV GZ-R株为美洲型高致病性毒株。试验结果可为进一步探索PRRSV在贵州省的流行趋势以及NSP2在PRRSV复制过程中的作用机制提供一定的参考。

关键词: 猪繁殖与呼吸综合征病毒(PRRSV); NSP2; 遗传变异分析

Abstract: In order to investigate the genetic variation of porcine reproductive and respiratory syndrome virus (PRRSV) strain GZ-R and the characteristics of NSP2 gene,in this study,two specific primers were designed based on NSP2 gene for nested RT-PCR,cloning and sequence analysis of the target gene.The results showed that the second round of nested PCR not only amplified the expected band of about 3 000 bp,but also amplified another band of 1 200 bp.The two DNA fragments were cloned into the pMD19-T vector,and two fragments were obtained by sequencing.The large fragment was 2 980 bp long and named as GZR-NSP2-L.The small fragment was 1 131 bp long and named as GZR-NSP2-S.The homology comparison results of the two fragments showed that GZR-NSP2-S had GZR-NSP2-L N-terminal nucleotide 1-945 and C-terminal 2 795-2 980 nucleotide,and lack of nucleotide between bits 946-2 794 in GZR-NSP2-L.The nucleotide homology of GZR-NSP2-L with VR-2332 strain and Lelystad virus strain was 81.2% and 48.6%.The results of homology comparison of encoded amino acids were consistent with the results of nucleotide homology,revealing that the PRRSV GZ-R strain belonged to the American-type strain.Comparison of amino acid deletion sites revealed that GZ-R NSP2 had 30 discontinuous amino acid deletions at positions 481 and 533-561.The deletion sites were consistent with PRRSV highly pathogenic strains.In summary,the PRRSV GZ-R strain could be identified as American-type highly pathogenic strain.The experimental results could provide some references for further exploring the epidemic trend of PRRSV in Guizhou province and the mechanism of NSP2 in PRRSV replication.

Key words: porcine reproductive and respiratory syndrome virus (PRRSV); NSP2; genetic variation analysis

中图分类号:

S855.3

张喜懿, 温贵兰, 管国丹, 陈广, 田浪, 杨佰启. 猪繁殖与呼吸综合征病毒贵州株GZ-R NSP2基因的克隆与遗传变异分析[J]. 中国畜牧兽医, 2020, 47(11): 3659-3666.

ZHANG Xiyi, WEN Guilan, GUAN Guodan, CHEN Guang, TIAN Lang, YANG Baiqi. Cloning and Genetic Variation Analysis of NSP2 Gene of Porcine Reproductive and Respiratory Syndrome Virus Guizhou Strain GZ-R[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3659-3666.

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猪繁殖与呼吸综合征病毒贵州株GZ-R NSP2基因的克隆与遗传变异分析

Cloning and Genetic Variation Analysis of NSP2 Gene of Porcine Reproductive and Respiratory Syndrome Virus Guizhou Strain GZ-R

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