Isolation,Identification of a Bovine viral diarrhea virus Strain, and Eukaryotic Expression of Its Erns Protein

doi:10.7668/hbnxb.20195598

Abstract

Abstract:

In order to isolate Bovine viral diarrhea virus(BVDV)from positive fecal samples and further achieve the expression of BVDV-Erns protein.This study utilized laboratory-preserved positive fecal samples,detected pathogens through qPCR,isolated and cultured the virus in MDBK cells,amplified the 5'-UTR region by RT-PCR,and verified the virus culture status using immunofluorescence technology.Constructing a phylogenetic tree based on 5'-UTR sequences using MEGA 11.0 software;expressing the BVDV-Erns protein through the baculovirus system in insects,and detecting protein expression with a His-tag monoclonal antibody by Western Blot.The results indicated that the BVDV strain isolated from feces,after cultivation,showed fluorescence signals by immunofluorescence,and RT-PCR amplification yielded a 297 bp specific fragment,consistent with the theoretical fragment size;the homology of the 5'-UTR sequence with the BVDV-1 reference strain reached 99%.The SDS-PAGE results showed a distinct band at 44-58 ku, and Western Blot verification with His tag antibody presented a specific reactive band,indicating successful expression of the BVDV-Erns protein.The study successfully isolated a BVDV-1 strain from feces,named BVDV-KMZ,and its 5'-UTR genetic characteristics are highly consistent with the BVDV-1 type.Additionally,the BVDV-Erns protein was expressed using the baculovirus expression system,and protein expression was confirmed by Western Blot.This lays the foundation for further establishment of serological detection methods,vaccine development,and research into the functions of the BVDV-Erns protein.

Key words: Bovine viral diarrhea virus(BVDV), Virus isolation, Baculovirus expression, Erns protein

CLC Number:

S823

ZHAO Kaiwei, PENG Yong, ZUO Weidong, WU Houyan, REN Miao, YIN Shanglian, ZHU Weijing, LANG Lixin, JI Xincheng, SONG Zhigang, AI Jun, XIN Jige. Isolation,Identification of a Bovine viral diarrhea virus Strain, and Eukaryotic Expression of Its Erns Protein[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(S1): 300-306. doi: 10.7668/hbnxb.20195598.

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Figures/Tables 11

Tab.1 Primers of BVDV 5'-UTR,BVDV-Erns and M13

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')	扩增片段 Amplified fragment	酶切位点 Restriction site
5'-UTR	F:CGAAGGCCGAAAAGA	297
	R:GCCATGTACAGCAGAGATT
BVDV-Erns	F:CCGGAATTCCAGAAAACATAACACAGTGGAACC	681	EcoR Ⅰ
	R:CCCAAGCTTAGCGTATGCTCCAAACCAC		Hind Ⅲ
M13	F:GTTTTCCCAGTCACGAC
	R:CAGGAAACAGCTATGAC

Tab.2 The qPCR primers of BVDV

基因 Gene	引物序列(5'-3') Primer sequence(5'-3')
BVDV	F:GRAGTCGTCARTGGTTCGAC
	R:TCAACTCCATGTGCCATGTAC
	p:famtgcyaygtggacgacggcatgctamra3

Fig.1 The qPCR identification results of feces samples 1.Negative control;2.BVDV NADL strain;3.Feces sample 1;4.Feces sample 2.

Fig.2 The results of RT-PCR on the supernatant of P6 cells M.DL2000 Marker;1-8.P6 cell supernatant;9.Negative control.

Fig.3 The result of BVDV-KMZ indirect immunofluorescence identification A.Negative control;B.NADL strain;C.BVDV-KMZ strain.

Fig.4 The evolutionary tree of BVDV-KMZ 5'-UTR sequences

Fig.5 Cloning and identification of BVDV-KMZ-Erns A.Amplified products of Erns from BVDV-KMZ strain: M. DL2000 Marker, 1-2.PCR amplified products of the BVDV Erns gene; B.Gel extraction products of the Erns amplified products: M.DL2000 Marker,1-2.Gel extraction products of the Erns PCR products from BVDV-KMZ strain; C.Bacterial liquid PCR of pMD18T-BVDV-Erns: M. DL2000 Marker, 1-5.Bacterial liquid PCR products of pMD18T-BVDV-Erns.

Fig.6 Enzyme digestion identification of pFastBacHTB-BVDB-Erns M. DNA Marker; 1-4. Different restriction enzyme digestion products of pFastBacHTB-BVDB-Erns.

Fig.7 Bacmid-BVDV-Erns verified by M13 primers M.DNA Marker;1-4.Products of Bacmid-BVDV-Erns amplified by M13 primer.

Fig.8 The rescue results of the recombinant virus A.Normal sf9 cells;B.Transfected sf9 cells for 96 h;C.Fluorescence of transfected cells for 24 h;D.Fluorescence of transfected cells for 96 h.

Fig.9 SDS-PAGE and Western Blot analysis of BVDV-Erns gene expression products A.SDS-PAGE analysis of the expression of BVDV-Erns protein;B.Western Blot analysis of BVDV-Erns protein expression.1,2.Cell supernatant lysate of the transfected Bacmid-BVDV-Erns sf9 cells.

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