新型鹅星状病毒 AH-2021 株的分离鉴定及其VP27-VP34蛋白多克隆抗体的制备

doi:10.7668/hbnxb.20195322

华北农学报 ›› 2025, Vol. 40 ›› Issue (2): 210-217. doi: 10.7668/hbnxb.20195322

所属专题： 植物保护；畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

新型鹅星状病毒 AH-2021 株的分离鉴定及其VP27-VP34蛋白多克隆抗体的制备

殷冬冬¹^,², 朱星星³, 兰梦蝶⁴, 彭梦玲³, 尹磊¹^,², 戴银¹^,², 沈学怀¹^,², 王洁茹¹^,², 赵瑞宏¹^,², 潘孝成¹^,²^,

¹ 安徽省农业科学院畜牧兽医研究所,安徽省畜禽疫病研究中心,安徽合肥 230001
² 安徽省畜禽产品重点实验室,安徽合肥 230001
³ 安徽农业大学动物科技学院,安徽合肥 230031
⁴ 宁国市动物卫生监督所,安徽宁国 242300

收稿日期:2024-10-29 出版日期:2025-04-28
通讯作者:
潘孝成(1971—),男,安徽霍邱人,副研究员,博士,主要从事畜禽病原微生物致病机制研究。
作者简介:
殷冬冬(1990—),男,安徽滁州人,助理研究员,博士,主要从事畜禽传染病防控研究。
基金资助:
安徽省农业科学院科研平台项目(2024YL016); 安徽省现代农业产业技术体系(AHCYJXTX-05-13)

Isolation and Identification of Novel Goose astrovirus AH-2021 Strain and Polyclonal Antibody Preparation of VP27-VP34 Protein

YIN Dongdong¹^,², ZHU Xingxing³, LAN Mengdie⁴, PENG Mengling³, YIN Lei¹^,², DAI Yin¹^,², SHEN Xuehuai¹^,², WANG Jieru¹^,², ZHAO Ruihong¹^,², PAN Xiaocheng¹^,²^,

¹ Livestock and Poultry Epidemic Diseases Research Center of Anhui Province,Institute of Animal Husbandry and Veterinary Science,Anhui Academy of Agricultural Sciences,Hefei 230001,China
² Anhui Provincial Key Laboratory of Livestock and Poultry Product,Hefei 230001,China
³ College of Animal Science and Technology,Anhui Agricultural University,Hefei 230031,China
⁴ Ningguo City Animal Health Supervision Institute,Ningguo 242300,China

Received:2024-10-29 Published:2025-04-28

摘要/Abstract

摘要：

为分析安徽六安地区新型鹅星状病毒(GAstV)的变异情况并表达其VP27-VP34蛋白,从六安某养殖场采集鹅痛风病料,经RT-PCR检测为阳性后,首先对鹅胚传代培养分离病毒,其次对分离毒株进行动物回归试验、全基因组扩增测序及遗传进化分析;随后,将分离株的VP27-VP34序列克隆至pCold-TF载体进行诱导表达,并将纯化后的重组蛋白免疫6 周龄雌性 BALB/c 小鼠制备多克隆抗体。通过间接免疫荧光(IFA)检测多克隆抗体特异性,利用琼脂扩散法检测血清抗体效价。结果表明,从临床病料样本中分离到 1 株鹅星状病毒,命名为 AH-2021 株。动物回归试验可见雏鹅心脏、肝脏表面尿酸盐沉积明显,肾脏发白、肿胀。遗传进化结果显示, AH-2021 株属于GAstV-1,与GenBank中的其他GAstV-1相似性为98.0%~99.0%。重组表达载体pCold-TF-VP27-VP34经 IPTG 诱导获得了目的蛋白,SDS-PAGE显示,重组蛋白分子质量约为110 ku,主要以上清形式存在。IFA试验结果显示,制备的多克隆抗体能够特异性识别新型鹅星状病毒,琼脂扩散结果显示,抗体效价可达 1:16。综上所述,从临床痛风雏鹅中分离鉴定到 1 株新型鹅星状病毒 AH-2021 株,动物回归试验表明,新型鹅星状病毒是导致雏鹅痛风的病原,制备了VP27-VP34 融合蛋白的多克隆抗体。

关键词: 新型鹅星状病毒, VP27-VP34 融合蛋白, 分离鉴定, 原核表达, 多克隆抗体

Abstract:

To analyze the variability of the novel Goose astrovirus(GAstV)in Lu'an,Anhui Province and to express the VP27-VP34 fusion protein,the gout samples were collected from a farm in Lu'an.After confirming positivity via RT-PCR,the virus was isolated by passage culture in goose embryos.Then,the isolated strain was subjected to an animal regression test,whole genome amplification sequencing and genetic evolution analysis.Subsequently,the VP27-VP34 fusion protein of the isolated strain was induced and expressed,and purified recombinant protein was used to immunize 6-week-old female BALB/c mice to produce polyclonal antibodies.Serum antibody titers were assessed using agar diffusion methods,and the specificity of the polyclonal antibodies was detected by indirect immunofluorescence(IFA).The specificity of the antibody was detected by indirect immunofluorescence(IFA),and the titer of the prepared antibody was detected by the agar diffusion method.The results showed that one strain of GAstV,named AH-2021 strain,was isolated from clinical samples.The animal regression test showed obvious urate deposition on the surface of the heart and liver of goslings,and the kidney was white and swollen.Genetic evolution results revealed that AH-2021 belonged to GAstV-1,showing 98.0%—99.0% identity with other GAstV-1 strains in GenBank.The recombinant expression vector pCold-TF-VP27-VP34 was induced by IPTG to obtain the target protein,and SDS-PAGE showed that the molecular weight of the recombinant protein was about 110 ku,which mainly existed in the form of supernatant.IFA results showed that the polyclonal antibody was able to specifically recognize the GAstV,and the agar diffusion results showed that the titer of polyclonal antibody was up to 1:16.In conclusion,a strain of novel GAstV AH-2021 was isolated from gouty goslings,and animal regression tests showed that the novel Goose astrovirus was the pathogen causing gout in goslings,and a polyclonal antibody to the VP27-VP34 fusion protein was prepared.

Key words: Novel Goose astrovirus, VP27-VP34 fusion protein, Isolation and identification, Prokaryotic expression, Polyclonal antibody

殷冬冬, 朱星星, 兰梦蝶, 彭梦玲, 尹磊, 戴银, 沈学怀, 王洁茹, 赵瑞宏, 潘孝成. 新型鹅星状病毒 AH-2021 株的分离鉴定及其VP27-VP34蛋白多克隆抗体的制备[J]. 华北农学报, 2025, 40(2): 210-217. doi: 10.7668/hbnxb.20195322.

YIN Dongdong, ZHU Xingxing, LAN Mengdie, PENG Mengling, YIN Lei, DAI Yin, SHEN Xuehuai, WANG Jieru, ZHAO Ruihong, PAN Xiaocheng. Isolation and Identification of Novel Goose astrovirus AH-2021 Strain and Polyclonal Antibody Preparation of VP27-VP34 Protein[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(2): 210-217. doi: 10.7668/hbnxb.20195322.

http://www.hbnxb.net/CN/Y2025/V40/I2/210

图/表 13

表1 新型鹅星状病毒检测引物

Tab.1 Primers for identification of novel Goose astrovirus

引物名称 Primer name	引物序列(5'—3') Primer sequence (5'—3')	产物大小/bp Product length
GAstV-F	AGAAGGTGCGGAAGAGTGGTATGA	300
GAstV-R	GCGAAGAGTGCGTAAGAGGTTGT
GAstV-1-F	AAACAGCGATATGGCGGC	845
GAstV-1-R	TGTACGACTTAGTCCAAGGAACG
GAstV-2-F	CACAGAGGCTACAATGCT	1 306
GAstV-2-R	CCTTCAACAACGACAATGG
GAstV-3-F	CAGTCCCTGTACAGATTTTA	1 366
GAstV-3-R	TCAACTTGTTCATCCTTTAC
GAstV-4-F	AGATTGATGAAGCCATTGAG	1 355
GAstV-4-R	CAGCCCGCCGTTCTGTCTGT
GAstV-5-F	AGGCTGTATCAGATATTGAT	1 278
GAstV-5-R	TCATTTTGTCATTAACGGG
GAstV-6-F	TCTGGTGAGTGGCGGACCGA	1 499
GAstV-6-R	TGTCATAACAGCCCACCAATTGTGT
GAstV-7-F	ACAACTGGACAAGGTACC	1 121
GAstV-7-R	TTTGCGGATTTTAAATGC

表2 鹅星状病毒参考株信息

Tab.2 Goose astrovirus reference strain information

毒株 Strains	GenBank登录号 GenBank accession No.	宿主 Host	时间 Year	地区 Area
CXZ	MH807626	鹅	2018	中国
SDXT	MN399857	鹅	2019	中国
SDPY	MH052598	鹅	2017	中国
AUAH4	MN428644	鹅	2018	中国
GD	MG934571	鹅	2017	中国
JSHA	MK125058	鹅	2019	中国
GTF-04	MN068023	鹅	2020	中国
SD01	MF772821	鹅	2017	中国
HNKF	MW592377	鹅	2020	中国
SL1	KF753804	鹅	2014	中国
TX/00	EU143850	火鸡	2012	中国
AAstV-3	AF206663	鹅	2012	中国
TAstV-2	NC_005790	火鸡	2018	中国
C-NGB	NC_012437	鸭	2018	中国
HB2015	KY646154	鹅	2015	中国
CAstV 2016	KY038163	鹅	2016	中国
CAstV 2018	MN725025	鹅	2018	中国
FLX	KY271027	鹅	2017	中国
AHDY	MH410610	鹅	2018	中国
AAstV-1	Y15936	鹅	2012	中国
G-4260	BAA92848	鹅	2006	中国
ANV 2017	MN732559	鹅	2017	中国
ANV	MG846415	鹅	2018	中国
HAstV SHI	FJ375759	人	2011	中国

图1 病原鉴定结果 A.发病雏鹅剖检变化;B.RT-PCR 检测结果:M.DL2000 DNA Marker,1.病例检测结果。

Fig.1 The results of pathogen identification A.Pathological changes of diseased goslings; B.The result of RT-PCR test:M.DL2000 DNA Marker,1.Test results for case.

图2 GAstV的分离鉴定 A.GAstV接种第3代鹅胚病变:左边为对照组,右边为试验组;B.RT-PCR 鉴定传代鹅胚的GAstV:M.DL2000 DNA Marker, 1.阴性对照, 2.阳性对照, 3—5.第1代到第3代鹅胚尿囊液。

Fig.2 Isolation and identification of GAstV A.Pathological changes of the 3rd generation goose embryos with GastV:the left is the control group and the right is the experimental group;B.Identification of GAstV by RT-PCR:M.DL2000 DNA Marker, 1.Negative control, 2.Positive control, 3—5.The 1st to 3rd generation of goose embryo allantoic fluid.

图3 雏鹅攻毒剖检病变 A.攻毒组心脏、肝脏尿酸盐沉积;B.攻毒组肾脏发白、肿胀;C.对照组心脏、肝脏无变化;D.对照组肾脏无变化。

Fig.3 Lesions of goslings after challenge A.Uate deposits in heart and liver in challenge group; B.Whitening and swelling in challenge group;C.No changes in heart and liver of control group; D.No changes in control kidney.

图4 雏鹅肾脏和脾病理组织学变化 A.攻毒组肾脏;B.攻毒组脾脏;C.对照组肾脏;D.对照组脾脏。

Fig.4 Pathological histological changes in kidney and spleen A.Kidney in challenge group;B.Spleen in challenge group; C.Kidney of control group;D.Spleen of control group.

图5 AH-2021株全基因组RT-PCR扩增结果 M.DL2000 DNA Marker;1.GAstV-1;2.GAstV-2;3.GAstV-3;4.GAstV-4;5.GAstV-5;6.GAstV-6;7.GAstV-7.

Fig.5 RT-PCR results of whole genome fragments of AH-2021 strain

图6 鹅星状病毒分离株基因组遗传进化分析

Fig.6 Genomic genetic evolution analysis of Goose astrovirus isolates

图7 鹅星状病毒分离株基因组同源性比对分析

Fig.7 Genome homology analysis results of Goose astrovirus isolates

图8 融合蛋白 VP27-VP34 原核表达 A.融合蛋白 VP27-VP34 诱导表达的SDS-PAGE分析:M.蛋白质Marker,1.未诱导的 pCold-TF,2.诱导的 pCold-TF,3.未诱导的 pCold-TF-VP27-VP34,4.诱导的 pCold-TF-VP27-VP34,5.细胞上清,6.包涵体;B.融合蛋白 VP27-VP34的纯化:M.蛋白质 Marker,1.纯化后 VP27-VP34。

Fig.8 Prokaryotic expression of the VP27-VP34 protein A.SDS-PAGE analysis of the fusion protein VP27-VP34 after induction expression:M.Protein Marker, 1.Uninduced pCold-TF,2.Ininduced pCold-TF,3.Uninduced pCold-TF-VP27-VP34,4.Induced pCold-TF-VP27-VP34,5.Cell supernatant,6.Inclusion body; B.Purification of the fusion protein VP27-VP34:M.Protein Marker,1.Purified VP27-VP34 protein.

图9 Western Blot 鉴定结果 M.蛋白分子质量标准;1.VP27-VP34 融合蛋白;2.pCold-TF 空载体。

Fig.9 The result of Western Blot assay M.Protein Marker; 1.VP27-VP34 protein; 2.pCold-TF vector.

图10 琼脂扩散法测抗体效价 1.PBS;2.未稀释小鼠血清;3—6.稀释2~16 倍的小鼠血清。

Fig.10 Antibody titer measured by the agar diffusion method 1.PBS; 2.Undiluted mouse serum; 3—6.Mouse serum diluted by 2—16 times.

图11 间接免疫荧光鉴定结果

Fig.11 The result of indirect immunofluorescence identification assay

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新型鹅星状病毒 AH-2021 株的分离鉴定及其VP27-VP34蛋白多克隆抗体的制备

Isolation and Identification of Novel Goose astrovirus AH-2021 Strain and Polyclonal Antibody Preparation of VP27-VP34 Protein

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