一株牛病毒性腹泻病毒的分离鉴定及其Erns蛋白的真核表达

doi:10.7668/hbnxb.20195598

华北农学报 ›› 2025, Vol. 40 ›› Issue (S1): 300-306. doi: 10.7668/hbnxb.20195598

所属专题： 植物保护；畜牧；

• 畜牧·水产·兽医 • 上一篇下一篇

一株牛病毒性腹泻病毒的分离鉴定及其Erns蛋白的真核表达

赵凯薇¹, 彭勇², 左卫东³, 吴后艳⁴, 任苗⁵, 尹尚莲⁵, 朱玮璟⁵, 郎立新⁵, 季新成⁶, 宋志刚⁶, 艾军⁵, 信吉阁¹

¹ 云南农业大学动物医学院,云南昆明 650201
² 瑞丽海关综合技术中心,云南瑞丽 678400
³ 勐腊海关综合技术中心,云南勐腊 666300
⁴ 昆明学院,云南昆明 650214
⁵ 昆明海关技术中心,云南昆明 650228
⁶ 海关总署国际检验检疫标准与技术法规研究中心,北京 101500

收稿日期:2024-11-20 出版日期:2025-12-29
通讯作者:
信吉阁(1981-),女,吉林桦甸人,教授,博士,主要从事基因表达与调控研究。
艾军(1977-),男,山西柳林人,研究员,博士,主要从事出入境动物检疫研究。
作者简介:
赵凯薇(1999-),女,河北邯郸人,在读硕士,主要从事动物疫病检测研究。
基金资助:
国家重点研发计划(2022YFC2601605); 云南省院士工作站(2018IC078); 兴滇英才支持计划“青年人才”专项(YNWR-QNBJ-2020-154)

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

ZHAO Kaiwei¹, PENG Yong², ZUO Weidong³, WU Houyan⁴, REN Miao⁵, YIN Shanglian⁵, ZHU Weijing⁵, LANG Lixin⁵, JI Xincheng⁶, SONG Zhigang⁶, AI Jun⁵, XIN Jige¹

¹ College of Veterinary Medicine, Yunnan Agricultural University,Kunming 650201,China
² Ruili Customs Comprehensive Technology Center,Ruili 678400,China
³ Mengla Customs Comprehensive Technology Center, Mengla 666300,China
⁴ Kunming University,Kunming 650214,China
⁵ Kunming Customs Technology Center,Kunming 650228,China
⁶ Research Center for International Inspection and Quarantine Standards and Technical Regulations,General Administration of Customs,Beijing 101500,China

Received:2024-11-20 Published:2025-12-29

摘要/Abstract

摘要：

为从阳性的粪便样品中分离牛病毒性腹泻病毒(BVDV),并进一步实现BVDV-Erns蛋白的表达。将实验室保存的BVDV阳性粪便样本,经qPCR检测病原,MDBK细胞分离培养病毒后,通过RT-PCR扩增5'-UTR区域,结合免疫荧光技术验证病毒培养情况;通过MEGA 11.0软件基于5'-UTR序列构建进化树;通过昆虫杆状病毒系统表达BVDV-Erns蛋白,Western Blot以His标签单抗检测蛋白表达。结果表明,从粪便中分离的BVDV毒株经培养后,免疫荧光显示出荧光信号,RT-PCR扩增获得297 bp特异性片段,与理论片段大小一致;5'-UTR序列与BVDV-1型参考株同源性达99%。SDS-PAGE检测结果在44~58 ku处见明显条带,Western Blot经His标签抗体验证呈现特异性反应条带,表明BVDV-Erns蛋白成功表达。该研究成功从粪便中分离出BVDV-1型毒株,命名为BVDV-KMZ,且其5'-UTR区域遗传特征与BVDV-1型高度一致,同时利用昆虫杆状病毒系统表达BVDV-Erns蛋白,Western Blot证实蛋白表达。为进一步建立血清学检测方法、疫苗研发及BVDV-Erns蛋白功能研究奠定基础。

关键词: 牛病毒性腹泻病毒, 病毒分离, 杆状病毒表达, Erns蛋白

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

中图分类号:

S823

赵凯薇, 彭勇, 左卫东, 吴后艳, 任苗, 尹尚莲, 朱玮璟, 郎立新, 季新成, 宋志刚, 艾军, 信吉阁. 一株牛病毒性腹泻病毒的分离鉴定及其Erns蛋白的真核表达[J]. 华北农学报, 2025, 40(S1): 300-306. doi: 10.7668/hbnxb.20195598.

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.

http://www.hbnxb.net/CN/Y2025/V40/IS1/300

图/表 11

表1 BVDV 5'-UTR、BVDV-Erns以及M13的引物

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

表2 BVDV qPCR引物

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

图1 粪便样品qPCR鉴定结果 1.阴性对照;2.BVDV NADL株;3.粪便样品1;4.粪便样品2。

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

图2 P6细胞上清液RT-PCR的结果 M.DL2000 Marker;1-8.P6细胞上清液;9.阴性对照。

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

图3 BVDV-KMZ 间接免疫荧光鉴定结果 A.阴性对照;B.NADL株;C.BVDV-KMZ株。

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

图4 BVDV-KMZ 5'-UTR序列进化树

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

图5 BVDV-KMZ-Erns的克隆、鉴定 A.BVDV-KMZ株Erns扩增产物:M. DL2000 Marker, 1-2.BVDV的Erns基因的PCR扩增产物;B.Erns扩增产物的胶回收产物:M.DL2000 Marker,1-2.BVDV-KMZ株ErnsPCR产物的胶回收产物;C.pMD18T-BVDV-Erns的菌液PCR:M.DL2000 Marker,1-5.pMD18T-BVDV-Erns的菌液PCR产物。

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.

图6 pFastBacHTB-BVDB-Erns酶切鉴定 M. DNA Marker; 1-4. 不同pFastBacHTB-BVDB-Erns酶切产物。

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

图7 M13引物验证Bacmid-BVDV-Erns M.DNA Marker;1-4.M13引物扩增Bacmid-BVDV-Erns产物。

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

图8 重组病毒的拯救结果 A.正常sf9细胞;B.转染96 h sf9细胞;C.转染24 h细胞荧光;D.转染96 h细胞荧光。

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.

图9 BVDV-Erns蛋白表达的SDS-PAGE及Western Blot分析 A.BVDV-Erns蛋白表达的SDS-PAGE分析;B.BVDV-Erns 蛋白表达的Western Blot分析。1,2.转染Bacmid-BVDV-Erns sf9细胞上清裂解液。

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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