Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

doi:10.3969/j.issn.1004-1524.2019.11.07

›› 2019, Vol. 31 ›› Issue (11): 1819-1824.DOI: 10.3969/j.issn.1004-1524.2019.11.07

• Animal Science • Previous Articles Next Articles

Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

ZHANG Kang¹, ZHANG Xuan², YAN Zunxiang¹, WANG Lei¹, ZHANG Kai¹, ZHANG Jingyan¹, LUO Yongjiang¹, QIU Zhengying¹, XUE Huan³, LI Jianxi^1,*

1. Key Laboratory of New Animal Drug Project of Gansu Province, Key Laboratory of Veterinary Drug Innovation of Ministry of Agriculture and Rural Affairs, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, China;
2. College of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China;
3. School of Basic Medical Science, Xi'an Jiaotong University, Xi'an 710061, China

Received:2019-04-20 Online:2019-11-25 Published:2019-12-04

Abstract

Abstract: In order to obtain the Core protein of bovine viral diarrhea virus (BVDV) and its polyclonal antibody, a pair of specific primers was designed and synthesized according to the coding sequence of the Core protein, and the BVDV Core gene was amplified by RT-PCR and inserted into the Pet30a vector. The recombinant plasmid Pet30a-Core was constructed and transformed into Escherichia coli TOP 10 competent cells by enzyme digestion. The positive recombinant bacteria were screened and the recombinant Core protein with molecular mass of 20 ku was induced to express with IPTG. The protein was purified by Ni column affinity method of the fusion protein, and the polyclonal antibody was prepared by immunizing New Zealand white rabbit with the purified recombinant protein, and the polyclonal antibody titer was determined by indirect ELISA to reach 1∶512 000. Western blot and indirect immunofluorescence assay (IFA) confirmed that polyclonal antibodies reacted with BVDV antigens in cells and had good immunogenicity and specificity. The rabbit polyclonal antibody against BVDV recombinant Core protein was successfully prepared, which laid a foundation for the detection of BVDV and the study of Core protein function.

Key words: bovine viral diarrhoea viruses (BVDV), Core protein, expression, polyclonal antibodies

CLC Number:

S858.23

ZHANG Kang, ZHANG Xuan, YAN Zunxiang, WANG Lei, ZHANG Kai, ZHANG Jingyan, LUO Yongjiang, QIU Zhengying, XUE Huan, LI Jianxi. Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies[J]. , 2019, 31(11): 1819-1824.

References

[1] YEŞILBAG K, ALPAY G, BECHER P. Variability and global distribution of subgenotypes of bovine viral diarrhea virus[J]. Viruses, 2017, 9(6): 128.
[2] BIANCHI M V, SILVEIRA S, MÓSENA A C S, et al. Pathological and virological features of skin lesions caused by BVDV in cattle[J]. Brazilian Journal of Microbiology, 2019, 50(1): 271-277.
[3] BRANZA-NICHITA N, DURANTEL D, CARROUEE-DURANTEL S, et al.Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers[J]. Journal of Virology, 2001, 75(8): 3527-3536.
[4] SEONG G, OEM J, LEE K, et al.Experimental infection of mice with bovine viral diarrhea virus[J]. Archives of Virology, 2015, 160(6): 1565-1571.
[5] EVANS C A, MOFFAT J L, HEMMATZADEH F, et al.The risk of transmission from sheep experimentally infected with BVDV-1c during the acute phase to BVDV naïve sheep[J]. Small Ruminant Research, 2017, 153: 5-8.
[6] HOUE H.Economic impact of BVDV infection in dairies[J]. Biologicals, 2003, 31(2): 137-143.
[7] NILNONT T, AIUMLAMAI S, KANISTANONT K, et al.Bovine viral diarrhea virus (BVDV) infection in dairy cattle herds in Northeast Thailand[J]. Tropical Animal Health and Production, 2016, 48(6): 1201-1208.
[8] TANAKA Y, SHIMOIKE T, ISHII K, et al.Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome[J]. Virology, 2000, 270(1): 229-236.
[9] MURRAY C L, MARCOTRIGIANO J, RICE C M.Bovine viral diarrhea virus core is an intrinsically disordered protein that binds RNA[J]. Journal of Virology, 2008, 82(3): 1294-1304.
[10] COLLEN T, CARR V, PARSONS K, et al.Analysis of the repertoire of cattle CD4+ T cells reactive with bovine viral diarrhoea virus[J]. Veterinary Immunology and Immunopathology, 2002, 87(3/4): 235-238.
[11] ELAHI S M, SHEN S, TALBOT B G, et al.Induction of humoral and cellular immune responses against the nucleocapsid of bovine viral diarrhea virus by an adenovirus vector with an inducible promoter[J]. Virology, 1999, 261(1): 1-7.
[12] TANAKA Y, SHIMOIKE T, ISHII K, et al.Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome[J]. Virology, 2000, 270(1): 229-236.
[13] NITAHARA-KASAHARA Y, FUKASAWA M, SHINKAI-OUCHI F, et al.Cellular vimentin content regulates the protein level of hepatitis C virus core protein and the hepatitis C virus production in cultured cells[J]. Virology, 2009, 383(2): 319-327.
[14] LI D, DONG H, LI S, et al.Hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus[J]. Journal of Virology, 2013, 87(10): 5707-5717.
[15] MCLAUCHLAN. Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes[J]. Journal of Viral Hepatitis, 2000, 7(1): 2-14.
[16] TELLINGHUISEN T L, RICE C M.Interaction between hepatitis C virus proteins and host cell factors[J]. Current Opinion in Microbiology, 2002, 5(4): 419-427.
[17] RAY R B, RAY R.Hepatitis C virus core protein: intriguing properties and functional relevance[J]. FEMS Microbiology Letters, 2001, 202(2): 149-156.
[18] 宫晓炜. 牛病毒性腹泻病毒感染形成和复制的分子机制研究[D]. 北京: 中国农业科学院, 2014.
GONG X W.Molecular mechanism of bovine viral diarrhea virus infection and replication[D]. Beijing: Chinese Academy of Agricultural Sciences, 2014.(in Chinese with English abstract)
[19] BALBÁS P. Understanding the art of producing protein and nonprotein molecules in Escherichia coli[J]. Molecular Biotechnology, 2001, 19(3): 251-268.
[20] LIU Z Q, XIA J, WANG G L, et al. Cloning and expression of the 4D8 gene from Hyalomma asiaticum tick[J]. Genetics and Molecular Research, 2016, 15(2):DOI: 10.4238/gmr.15027951.

Prokaryotic expression of Core protein of bovine viral diarrhoea viruses and preparation of polyclonal antibodies

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	ZHAO Xiuping, WANG Shuang, YAN Xingyi, DUAN Qiang, ZHANG Shuai, CHEN Yongsheng, LI Guorui. Expression, purification and bioinformatics analysis of Magnaporthe oryzae MGG-01005 [J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 470-478.
[2]	WU Junjing, QIAO Mu, ZHOU Jiawei, MEI Shuqi, PENG Xianwen. Function of porcine non-coding RNA lnc-000649 during PRRSV infection [J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 223-229.
[3]	HUANG Yongming, SONG Fang, WANG Ce, YAO Jinglei, WANG Zhijing, HE Ligang, WU Liming, JIANG Yingchun. Effects of root pruning on growth and expression of related genes in Poncirus trifoliata [J]. Acta Agriculturae Zhejiangensis, 2021, 33(2): 270-277.
[4]	HE Jiaqi, ZHAI Ying, ZHANG Jun, QIU Shuang, LI Mingyang, ZHAO Yan, ZHANG Meijuan, MA Tianyi. Cloning and expression analysis of GmDof1.5 in soybean under abiotic stress [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 1-7.
[5]	SU Xuesi, ZHANG Yubao, WANG Ruoyu, WANG Yajun, TANG Guoliang, JIN Weijie. Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of its polyclonal antibody [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 104-111.
[6]	LIU Jinyu, HUANG Ying. Prokaryotic expression and antibody preparation of full-length and N-terminal half of SpTrz2 protein in Schizosaccharomyces pombe [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 34-42.
[7]	WANG Weike, LU Na, YAN Jing, SONG Jiling, YUAN Weidong, ZHOU Zufa. Cloning and expression analysis of PpSAMS gene of Pleurotus pulmonarius [J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 62-68.
[8]	XU Xiuhong, LIU Jinliang, LI Dongcheng, LIU Renxiang. Analysis of nicotine content and related gene expression in different types of tobacco germplasm [J]. , 2020, 32(9): 1555-1563.
[9]	WANG Wei, GUN Shuangbao, WANG Pengfei, HUANG Xiaoyu, XIE Kaihui, LUO Ruirui, GAO Xiaoli, ZHANG Bo, YAN Zunqiang, YANG Qiaoli, MA Yanping. Tissue expression and significant target genes analysis of swine miR-204 [J]. , 2020, 32(9): 1564-1573.
[10]	LIU Kunju, ZHANG Xiaohui, PANG Youzhi, ZHAO Shujuan, QI Yanxia, WANG Qiankun. Relationship of plumage color with expression and polymorphism of GNAS gene in Korean quail [J]. , 2020, 32(8): 1369-1377.
[11]	SHENG Zhongwei, JI Gaige, LIU Yifan, JU Xiaojun, SHAN Yanju, ZOU Jianmin, ZHANG Ming, TU Yunjie, SHU Jingting. Study on mRNA expression pattern of IGF-1R gene in chicken skeletal muscles during early development [J]. , 2020, 32(7): 1160-1165.
[12]	HUANG Xiaozhen, QIAO Zhongquan, ZENG Huijie, LI Yongxin, HE Gang, WANG Xiaoming. Isolation and expression of floral organ development regulating gene LiFUL1 in Lagerstroemia indica L. [J]. , 2020, 32(7): 1206-1214.
[13]	SUN Ruiping, WANG Feng, CHAO Zhe, LIU Hailong, XING Manping, LIU Quanwei, HUANG Lili, ZHENG Xinli, WEI Limin. Cloning and tissue expression analysis of PDK4 gene in Tunchang pig [J]. , 2020, 32(6): 978-985.
[14]	LI Qiuling, QI Ying, WANG Chen, ZHANG Yiming, WANG Xinyu, SHANG Xiaolan, JIA Yonghong, LI Meiru, CHU Mingxing. Effect of heat stress on gene expressions and signaling pathways of mammary gland in Chinese Holstein [J]. , 2020, 32(5): 770-778.
[15]	CHEN Yunlu, SHAN Ying, LUO Hao, XU Jidong, ZHAO Lingyan, FANG Weihuan, LI Xiaoliang. Prokaryotic expression of porcine type Ⅲ interferon and studying on its antiviral activity [J]. , 2020, 32(5): 779-788.