犬冠状病毒N蛋白的原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2020.05.004

Abstract

Abstract: The purpose of this study was to clone the N gene of canine coronavirus(CCV),express the N protein in vitro,and prepare a polyclonal antibody against this protein for the diagnosis of CCV and its antigen detection.Referring to the N gene sequence of CCV in GenBank (accession No.:KY063618.2) and the N gene of CCV epidemic strain was selected,and the recombinant expression plasmid pET-B2M-N was constructed by codon optimization and gene synthesis.Finally,an effective gene was selected to construct the recombinant plasmid,which transformed into E.coli DH5α constructer.After sequencing,positive clones were transformed into E.coli BL21 (DE3) strain and induced by IPTG.Finally,IPTG concentration of 0.5 mmol/L was used to induce at 30 ℃.The results showed that the recombinant plasmid pET-B2M-N containing canine coronavirus N gene was successfully constructed.After optimizing the induction conditions,the recombinant protein with a size of about 49 ku was successfully expressed.The recombinant protein was mixed with Freund’s adjuvant to immunize Japanese White rabbits G767 and G768 for several times,and the G768 antibody titer was up to 1:512 000 assayed by indirect ELISA.The antibody could be purified to 10 mg/mL.The charactered of polyclonal antibodies detected by ELISA,Western blotting and immunofluorescence,indicating that the expressed recombinant N protein had good immunogenicity and reactogenicity.This study provides a basis for the detection of canine coronavirus antigen and antibodies and the establishment of target CCV diagnostic kit.

Key words: canine coronavirus(CCV); N gene; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺5

ZHOU Yahua, YIN Juanbin, LIU Le, YIN Xiangping, LI Yanmin, ZHANG Zhidong. Prokaryotic Expression of Canine Coronavirus N Protein and Preparation of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(5): 1318-1325.

References

[1] 熊炜,张强,王艳,等.犬冠状病毒核衣壳蛋白的原核表达及间接ELISA检测方法的建立[J].中国兽医杂志,2015,51(8):86-88. XIONG W,ZHANG Q,WANG Y,et al.Prokaryotic expression of nucleocapsid protein of canine coronavirus and establishment of indirect ELISA[J].Chinese Journal of Veterinary Medicine,2015,51(8):86-88.(in Chinese)
[2] 王静,梁琳,罗亚坤,等.犬冠状病毒BC-1株核衣壳蛋白的原核表达及生物信息学分析[J].畜牧兽医科技信息,2018,2:24-26. WANG J,LIANG L,LUO Y K,et al.Prokaryotic expression and bioinformatics analysis of nucleocapsid protein of canine coronavirus BC-1 strain[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2018,2:24-26.(in Chinese)
[3] WESSELING J G,VENNEMA H,GODEKE G J,et al.Nucleotide sequence and expression of the spike (S) gene of canine coronavirus and comparison with the S proteins of feline and porcine coronaviruses[J].Journal of General Virology,1994,75(Pt7):1789-1794.
[4] 胡桂学,夏咸柱,武银莲,等.犬冠状病毒的分子生物学研究进展[J].经济动物学报,1999,2:61-65. HU G X,XIA X Z,WU Y L,et al.Advances of molecular biology of canine coronavirus[J].Journal of Economic Animal,1999,2:61-65.(in Chinese)
[5] TAHARA S M,DIETLIN T A,BERGMANN C C,et al.Coronavirus translational regulation:Leader affects mRNA efficiency[J].Virology,1994,202(2):621-630.
[6] HAAN C A M D,ROTTIER P J M.Molecular interactions in the assembly of coronaviruses[J].Advances in Virus Research,2005,64:165-230.
[7] 张建武,陈燕军,吉荣,等.犬冠状病毒分子生物学研究进展[J].动物医学进展,2004,2:54-57. ZHANG J W,CHEN Y J,JI R,et al.Progress of research on molecular biology of canine coronavirus[J].Progress in Veterinary Medicine,2004,2:54-57.(in Chinese)
[8] 王珂,张守涛,郭亚楠.GFAP单克隆抗体制备及ELISA检测方法研究[J].郑州大学学报(理学版),2016,48(2):95-100. WANG K,ZHANG S T,GUO Y N.Preparation of Anti-GFAP monoclonal antibodies and development of double antibody sandwich ELISA for GFAP[J].Journal of Zhengzhou University(Natural Science Edition),2016,48(2):95-100.(in Chinese)
[9] CZERNY C P,EICHHORN W.Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus:Establishment of an efficient ELISA for antigen detection in feces[J].Veterinary microbiology,1989,20(2):111-122.
[10] SIMON N,SPERBER C,VOIGTLÄNDER C,et al.Improved stability of polyclonal antibodies:A case study with lyophilization-conserved antibodies raised against epitopes from the malaria parasite plasmodium falciparum[J].European Journal of Pharmaceutical Sciences,2020,142:105086.
[11] MCBRIDE R,VAN ZYL M,FIELDING B.The Coronavirus nucleocapsid is a multifunctional protein[J].Viruses,2014,6(8):2991-3018.
[12] WANG C,REN X.Preparation and characterization of polyclonal antibody against severe acute respiratory syndrome-associated coronavirus spike protein[J].Hybridoma,2010,29(6):511-516.
[13] MENG F,YIN J,LI X,et al.Production and characterization of a monoclonal antibody against spike protein of transmissible gastroenteritis virus[J].Hybridoma (Larchmt),2010,29(4):345-350.
[14] WANG X,LI H,LI Y,et al.Effect of porcine epidemic diarrhea virus nsp1 on type Ⅰinterferon response[J].Chinese Journal of Biotechnology,2017,33(8):1325.
[15] XUFANG D,ALBERT D X,VAN GEELEN A,et al.Coronavirus endoribonuclease activity in porcine epidemic diarrhea virus suppresses type Ⅰ and type Ⅲ interferon responses[J].Journal of Virology,2019,93(8):e02000-18.
[16] 李红杰,李永涛,刘延珂,等.猪流行性腹泻病毒Nsp7蛋白的表达与多克隆抗体制备[J].畜牧与兽医,2017,49(11):87-91. LI H J,LI Y T,LIU Y K,et al.Prokaryotic expression of Nsp7 of porcine epidemic diarrhea virus and preparation of polyclonal antibodies against Nsp7[J].Animal Husbandry & Veterinary Medicine,2017,49(11):87-91.(in Chinese)
[17] 李成,张雨迪,黄小波,等.猪丁型冠状病毒S1基因的克隆、原核表达及多克隆抗体制备[J].畜牧兽医学报,2018,49(6):1256-1264. LI C,ZHANG Y D,HUANG X B,et al.Cloning and prokaryotic expression of porcine deltacoronavirus S1 gene and preparation of polyclonal antibody[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(6):1256-1264.(in Chinese)
[18] 苏鹏,龚国利.优化大肠杆菌表达外源蛋白的研究进展[J].生物技术通报,2017,33(2):16-23. SU P,GONG G L.Research progress on optimizing the expression of exogenous proteins in Escherichia coli[J].Biotechnology Bulletin,2017,33(2):16-23.(in Chinese)
[19] 周芝海,谭耀荣,郑瑶瑶,等.猪急性腹泻综合征冠状病毒核衣壳蛋白的原核表达及其多克隆抗体的制备[J].中国兽医科学,2019,49(9):1179-1186. ZHOU Z H,TAN Y R,ZHENG Y Y,et al.Prokaryotic expression and polyclonal antibody preparation of swine acute diarrhea syndrome coronavirus nucleocapsid protein[J].Chinese Veterinary Science,2019,49(9):1179-1186.(in Chinese)
[20] JAIN T,KADAM M,JAIN A,et al.Prokaryotic expression of N gene of infectious bronchitis virus and assessment of its immunogenicity and antigenicity[J].Indian Journal of Animal Research,2013,47(4):292-300.

Prokaryotic Expression of Canine Coronavirus N Protein and Preparation of Its Polyclonal Antibody

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