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

doi:10.7668/hbnxb.20195322

Abstract

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

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.

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Figures/Tables 13

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

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

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.

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.

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.

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.

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

Fig.6 Genomic genetic evolution analysis of Goose astrovirus isolates

Fig.7 Genome homology analysis results of Goose astrovirus isolates

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.

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

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.

Fig.11 The result of indirect immunofluorescence identification assay

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