两株GI.1型和GI.2型兔出血症病毒RdRp基因的克隆与分析

doi:10.3969/j.issn.1004-1524.2023.06.06

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (6): 1286-1296.DOI: 10.3969/j.issn.1004-1524.2023.06.06

两株GI.1型和GI.2型兔出血症病毒RdRp基因的克隆与分析

庞雪晴¹(), 唐诗¹, 曾红梅¹, 赵位¹, 王印¹, 罗燕¹, 姚学萍¹, 任梅渗¹, 任永军², 杨泽晓¹^,^*()

1.四川农业大学动物医学院,四川成都 611130
2.四川省畜牧科学研究院,四川成都 610066

收稿日期:2022-05-23 出版日期:2023-06-25 发布日期:2023-07-04
通讯作者: *杨泽晓,E-mail : yzxyang2003@126.com
作者简介:庞雪晴(1998—),女,山东临沂人,硕士研究生,研究方向为动物疫病防治与兽医公共卫生学。E-mail :1209320301@qq.com
基金资助:
国家自然科学基金(31402222);四川农业大学学科建设双支计划项目(2021993257)

Cloning and analysis of RdRp gene in two strains of GI.1 and GI.2 RHDV

PANG Xueqing¹(), TANG Shi¹, ZENG Hongmei¹, ZHAO Wei¹, WANG Yin¹, LUO Yan¹, YAO Xueping¹, REN Meishen¹, REN Yongjun², YANG Zexiao¹^,^*()

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Sichuan Institute of Animal Husbandry Science, Chengdu 610066, China

Received:2022-05-23 Online:2023-06-25 Published:2023-07-04

摘要/Abstract

摘要：

为了解兔出血症病毒(rabbit hemorrhagic disease virus, RHDV)RdRp基因的遗传稳定性和变异规律,本研究分别克隆了RHDV SCH04株和RHDV2 SCCN03株的RdRp基因,然后对基因序列进行比对和系统发育分析,并通过编码蛋白质的理化性质、信号肽、跨膜区域、二级结构、三级结构、磷酸化和糖基化修饰位点的预测,对RdRp进行生物信息学分析。结果显示,两株RdRp基因序列全长均为1 548 bp,编码516个氨基酸。GI.1型RHDV毒株间序列相似性为86.9%~99.9%,GI.2型RHDV毒株间为91.3%~99.9%。GI.1型和GI.2型RHDV毒株间序列相似性为84.3%~88.3%。SCH04株和SCCN03株分别属于GI.1a型和GI.2型RHDV,且SCH04株和SCCN03株的RdRp基因核苷酸序列相似性为84.95%,其编码蛋白存在17个氨基酸差异位点,氨基酸序列相似性为96.71%;二者均属于稳定蛋白,不存在信号肽和跨膜区域;其分子量、等电点、脂溶系数、平均亲水系数和不稳定系数等指标无显著差异。SCH04株RdRp的α螺旋和β转角比例比SCCN03株略高,分别为41.86%和4.26%,而SCCN03株RdRp的延伸链和无规则卷曲比例比SCH04株略高,分别为12.79%和43.41%;SCCN03株 RdRp比SCH04株多4个磷酸化位点和1个N-糖基化位点。分析结果表明不同型间RHDV RdRp基因核苷酸变异率较高,GI.1 RHDV毒株间的变异范围大于GI.2 RHDV毒株。SCH04株和SCCN03株RHDV RdRp的氨基酸序列相似性高于核苷酸序列相似性,其二级结构和高级结构差异较小,遗传相对稳定。本研究为RHDV的遗传演变和生物合成等分子病原学研究提供了科学参考。

关键词: 兔出血症病毒, RdRp, 对比分析, 生物信息学分析

Abstract:

In order to understand the genetic stability and variation of RHDV RdRp gene, the RdRp gene of RHDV SCH04 strain and RHDV2 SCCN03 strain were cloned respectively in this study, then the gene sequences were compared and phylogenetic analysis was carried out. The bioinformatics analysis of RdRp was carried out by predicting the physical and chemical properties, signal peptides, transmembrane region, secondary structure, tertiary structure, phosphorylation and glycosylation modification sites of the coding protein. The results showed that the full length of RdRp gene sequences of the two strains were 1 548 bp, encoding 516 amino acids. The sequence similarity between GI.1 RHDV strains was 86.9%-99.9% and that between GI.2 RHDV strains was 91.3%-99.9%. The sequence similarity between GI.1 and GI.2 RHDV strains was 84.3-88.3%. SCH04 and SCCN03 strains belonged to GI.1a and GI.2 RHDV respectively, and the similarity of nucleotide sequence of RdRp gene between SCH04 and SCCN03 strains was 84.95%. There were 17 amino acid difference sites in coding protein, the amino acid sequence similarity was 96.71%. Both of them belong to stable proteins without signal peptides and transmembrane regions. There was no significant difference in molecular weight, isoelectric point, fat-soluble coefficient, average hydrophilic coefficient and instability coefficient. The proportions of α helix and β rotation of RdRp in SCH04 strain were slightly higher than those in SCCN03 strain (41.86% and 4.26%, respectively), while the proportions of extended chain and random curl in SCCN03 strain were slightly higher than those in SCH04 strain (12.79% and 43.41%, respectively). SCCN03 strain’s RdRp had 4 more phosphorylation sites and 1 more N-glycosylation site than SCH04 strain’s RdRp. The results showed that the nucleotide variation rate of RHDV RdRp gene among different types was large, and the variation range of GI.1 RHDV strain was larger than that of GI.2 RHDV strain. The amino acid sequence similarity of RHDV RdRp of SCH04 strain and SCCN03 strain is higher than that of nucleotide, the difference between secondary structure and advanced structure is small, and the inheritance is relatively stable. These results provide scientific reference for the molecular etiology of RHDV such as genetic evolution and biosynthesis.

Key words: rabbit hemorrhagic disease virus, RdRp, comparative analysis, bioinformatics analysis

中图分类号:

S858.291

庞雪晴, 唐诗, 曾红梅, 赵位, 王印, 罗燕, 姚学萍, 任梅渗, 任永军, 杨泽晓. 两株GI.1型和GI.2型兔出血症病毒RdRp基因的克隆与分析[J]. 浙江农业学报, 2023, 35(6): 1286-1296.

PANG Xueqing, TANG Shi, ZENG Hongmei, ZHAO Wei, WANG Yin, LUO Yan, YAO Xueping, REN Meishen, REN Yongjun, YANG Zexiao. Cloning and analysis of RdRp gene in two strains of GI.1 and GI.2 RHDV[J]. Acta Agriculturae Zhejiangensis, 2023, 35(6): 1286-1296.

图/表 9

参考文献 25

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GenBank序列号 GenBank No.	年份 Year	国家 Country	GenBank序列号 GenBank No.	年份 Year	国家 Country
U54983	1987	捷克Czech	KP144791	2004	波兰Poland
KP144790	1988	波兰Poland	KU882094	2004	波兰Poland
EF558578	1989	德国Germany	DQ280493	2005	中国China
Z29514	1989	法国France	KY319035	2005	波兰Poland
M67473	1989	德国Germany	EU003578	2005	美国America
KP144789	1989	波兰Poland	JF412629	2005	中国China
X87607	1989	意大利Italy	KY171748	2007	中国China
EU003579	1990	意大利Italy	EU871528	2007	澳大利亚
EF558575	1992	英国Britain	HM623309	2009	中国China
EF558576	1993	德国Germany	MN737114	2010	法国France
EF558577	1993	德国Germany	KM878681	2011	西班牙Spain
KU882093	1994	波兰Poland	KF677011	2012	波兰Poland
KP144792	1994	波兰Poland	KY319032	2013	波兰Poland
JX886002	1995	葡萄牙Portugal	KY679903	2013	波兰Poland
MT628287	1995	法国France	KY679904	2013	波兰Poland
EF558583	1996	德国Germany	KY679905	2014	波兰Poland
EF558586	1996	德国Germany	KY319031	2015	波兰Poland
EF558572	1996	德国Germany	KY437668	2016	中国China
DQ205345	1997	中国China	MF407653	2016	西班牙Spain
KU882092	1998	波兰Poland	MN061492	2016	荷兰Holland
AF258618	2000	美国America	MN786321	2016	法国France
EF558581	2000	德国Germany	MN853659	2018	波兰Poland
KU882095	2000	波兰Poland	MN853660	2018	波兰Poland
EU003581	2001	美国America	MN853661	2018	波兰Poland
EU003582	2001	美国America	MT434995	2020	中国China
DQ189077	2001	巴林Bahrain	MT505389	2020	中国China
GQ166866	2001	美国America	MT506234	2020	美国America
AB300693	2002	日本Japan	MT506237	2020	美国America
EF558584	2002	德国Germany	MT586027	2020	中国China
AY523410	2004	中国China	MW926383	2020	美国America
KY679902	2004	波兰Poland

GenBank序列号 GenBank No.	年份 Year	国家 Country	GenBank序列号 GenBank No.	年份 Year	国家 Country
U54983	1987	捷克Czech	KP144791	2004	波兰Poland
KP144790	1988	波兰Poland	KU882094	2004	波兰Poland
EF558578	1989	德国Germany	DQ280493	2005	中国China
Z29514	1989	法国France	KY319035	2005	波兰Poland
M67473	1989	德国Germany	EU003578	2005	美国America
KP144789	1989	波兰Poland	JF412629	2005	中国China
X87607	1989	意大利Italy	KY171748	2007	中国China
EU003579	1990	意大利Italy	EU871528	2007	澳大利亚
EF558575	1992	英国Britain	HM623309	2009	中国China
EF558576	1993	德国Germany	MN737114	2010	法国France
EF558577	1993	德国Germany	KM878681	2011	西班牙Spain
KU882093	1994	波兰Poland	KF677011	2012	波兰Poland
KP144792	1994	波兰Poland	KY319032	2013	波兰Poland
JX886002	1995	葡萄牙Portugal	KY679903	2013	波兰Poland
MT628287	1995	法国France	KY679904	2013	波兰Poland
EF558583	1996	德国Germany	KY679905	2014	波兰Poland
EF558586	1996	德国Germany	KY319031	2015	波兰Poland
EF558572	1996	德国Germany	KY437668	2016	中国China
DQ205345	1997	中国China	MF407653	2016	西班牙Spain
KU882092	1998	波兰Poland	MN061492	2016	荷兰Holland
AF258618	2000	美国America	MN786321	2016	法国France
EF558581	2000	德国Germany	MN853659	2018	波兰Poland
KU882095	2000	波兰Poland	MN853660	2018	波兰Poland
EU003581	2001	美国America	MN853661	2018	波兰Poland
EU003582	2001	美国America	MT434995	2020	中国China
DQ189077	2001	巴林Bahrain	MT505389	2020	中国China
GQ166866	2001	美国America	MT506234	2020	美国America
AB300693	2002	日本Japan	MT506237	2020	美国America
EF558584	2002	德国Germany	MT586027	2020	中国China
AY523410	2004	中国China	MW926383	2020	美国America
KY679902	2004	波兰Poland

毒株 Strain	分子式 Formula	分子量 Molecular weight/u	等电点 Theoretical pI	脂溶系数 Aliphatic index	平均亲水系数 Aliphatic index	不稳定系数 Instability index	正电荷(Arg+Lys)数 Number of negative charges (Arg+Lys)	负电荷(Asp+Glu)数 Number of negative charges (Asp+Glu)
SCH04	C₂₅₈₂H₄₀₆₅N₆₉₁O₇₅₅S₂₈	57 765.54	6.43	89.92	-0.126	37.00	62	65
SCCN03	C₂₅₇₇H₄₀₅₈N₆₉₄O₇₆₀S₂₉	57 852.50	6.33	88.97	-0.160	36.01	62	66

毒株 Strain	分子式 Formula	分子量 Molecular weight/u	等电点 Theoretical pI	脂溶系数 Aliphatic index	平均亲水系数 Aliphatic index	不稳定系数 Instability index	正电荷(Arg+Lys)数 Number of negative charges (Arg+Lys)	负电荷(Asp+Glu)数 Number of negative charges (Asp+Glu)
SCH04	C₂₅₈₂H₄₀₆₅N₆₉₁O₇₅₅S₂₈	57 765.54	6.43	89.92	-0.126	37.00	62	65
SCCN03	C₂₅₇₇H₄₀₅₈N₆₉₄O₇₆₀S₂₉	57 852.50	6.33	88.97	-0.160	36.01	62	66

两株GI.1型和GI.2型兔出血症病毒RdRp基因的克隆与分析

Cloning and analysis of RdRp gene in two strains of GI.1 and GI.2 RHDV

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