猪圆环病毒3型新疆株Cap蛋白的原核表达及纯化

doi:10.16431/j.cnki.1671-7236.2020.08.036

Abstract

Abstract: The purpose of this study was to prepare porcine circovirus type 3 (PCV3) capsid protein by construct a prokaryotic expression vector pGEX-4T-1-Cap.Recombinant plasmid pGEX-4T-1-Cap was constructed by inserting codon optimized PCV3 Cap gene into pGEX-4T-1 vector,and then transformed into Escherichia coli BL21 (DE3) competent cell.The optimal induction conditions were screened out,and the recombinant Cap protein was purified by glutathione agarose resin.The results showed that the recombinant plasmid pGEX-4T-1-Cap was successfully constructed.A large amount of soluble recombinant capsid protein could be obtained under the condition that the final concentration of IPTG was 0.1 mmol/L and induced at 16 ℃ for 20 h.SDS-PAGE results showed that the molecular mass of the recombinant capsid protein was 51.6 ku with the same size as expected.Western blotting analysis showed that the purified Cap protein reacted positively with mouse anti-GST monoclonal antibody and PCV3 rabbit-derived positive serum,further confirming the expression of the recombinant protein.Therefore,the recombinant protein PCV3 Cap expressed and purified in this study,it laid a foundation for the development of new gene engineering subunit vaccine and the establishment of ELISA method.

Key words: porcine circovirus type 3(PCV3); capsid protein; prokaryotic expression; protein purification

CLC Number:

S855.3

GU Siying, QU Yonggang, WEI Qi, WU Yuanyuan, YU Huiju, LI Yan. Prokaryotic Expression and Purification of Cap Protein of Xinjiang Porcine Circovirus Type 3 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2660-2665.

References

[1] TISCHER I,GELDERBLOM H,VETTERMANN W,et al.A very small porcine virus with circular single-stranded DNA[J].Nature,1982,295(5844):64-66.
[2] TISCHER I,RASCH R,TOCHTERMANN G.Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines[J].Zentralblatt fur Bakteriologie,Parasitenkunde,Infektionskrankheiten und Hygiene.Erste Abteilung Originale.Reihe A:Medizinische Mikrobiologie und Parasitologie,1974,226(2):153-167.
[3] RACHEL P,PABLO P,SHANG P C,et al.A novel porcine circovirus distantly related to known circoviruses is associated with porcine dermatitis and nephropathy syndrome and reproductive failure[J].Journal of Virology,2016,91(1):1816-1879.
[4] PHAN T G,GIANNITTI F,ROSSOW S,et al.Detection of a novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation[J].Virology Journal,2016,13(1):184-197.
[5] WANG W,SUN W,CAO L,et al.An epidemiological investigation of porcine circovirus 3 infection in cattle in Shandong province,China[J].BMC Veterinary Research,2019,15(1):60-68.
[6] KEDKOVID R,WOONWONG Y,ARUNORAT J,et al.Porcine circovirus type 3(PCV3) infection in grower pigs from a Thai farm suffering from porcine respiratory disease complex (PRDC)[J].Veterinary Microbiology,2018,215:71-76.
[7] JIANG H,WEI L,WANG D,et al.ITRAQ-based quantitative proteomics reveals the first proteome profiles of piglets infected with porcine circovirus type 3[J].Journal of Proteomics,2020,212:103598.
[8] 湛洋,王东亮,王乃东,等.猪圆环病毒3型检测及其Cap结构序列和抗原性预测分析[J].畜牧兽医学报,2017,48(6):1076-1084. ZHAN Y,WANG D L,WANG N D,et al.Detection of porcine circovirus type 3 and its Cap structure sequence and antigenicity prediction analysis[J].Acta Veterinary et Zootechnica Sinica,2017,48(6):1076-1084.(in Chinese)
[9] KU X,CHEN F,LI P,et al.Identification and genetic characterization of porcine circovirus type 3 in China[J].Transboundary & Emerging Diseases,2017,64(3):703-708.
[10] FRANZO G,LEGNARDI M,HJULSAGER C K,et al.Full-genome sequencing of porcine circovirus 3 field strains from Denmark,Italy and Spain demonstrates a high within-Europe genetic heterogeneity[J].Transboundary & Emerging Diseases,2018,65(3):602-606.
[11] KIM H R,PARK Y R,LIM D R,et al.Multiplex Real-time polymerase chain reaction for the differential detection of porcine circovirus 2 and 3[J].Journal of Virological Methods,2017,250:11-16.
[12] YE X,BERG M,FOSSUM C,et al.Detection and genetic characterisation of porcine circovirus 3 from pigs in Sweden[J].Virus Genes,2018,54(3):466-469.
[13] BERA B C,CHOUDHARY M,ANAND T,et al.Detection and genetic characterization of porcine circovirus 3(PCV3) in pigs in India[J].Transboundary and Emerging Diseases,2019,6:11-18.
[14] FACCINI S,BARBIERI I,GILIOLI A,et al.Detection and genetic characterization of porcine circovirus type 3 in Italy[J].Transboundary & Emerging Diseases,2017,64(6):1661-1664.
[15] VARGAS D,CAMPOS F S,BONIL L,et al.First detection of porcine circovirus type 3 in Colombia and the complete genome sequence demonstrates the circulation of PCV3a1 and PCV3a2[J].Veterinary Medicine and Science,2019,5(2):182-188.
[16] YUZHAKOV A G,RAEV S A,ALEKSEEV K P,et al.First detection and full genome sequence of porcine circovirus type 3 in Russia[J].Virus Genes,2018,54(4):608-611.
[17] SHIZUKA H,YOSHIYUKI O,YOSHIAKI F,et al.First detection of porcine circovirus type 3 in Japan[J].The Journal of Veterinary Medical Science,2018,80(9):1468-1472.
[18] STADEJEK T,WOŹNIAK A,MILEK D,et al.First detection of porcine circovirus type 3 on commercial pig farms in Poland[J].Transboundary & Emerging Diseases,2017,65(5):1350-1353.
[19] QI S S,SU M J,GUO D H,et al.Molecular detection and phylogenetic analysis of porcine circovirus type 3 in 21 provinces of China during 2015-2017[J].Transboundary and Emerging Disease,2019,66(2):1004-1015.
[20] OUYANG T,NIU G,LIU X,et al.Recent progress on porcine circovirus type 3[J].Infection,Genetics and Evolution,2019,73:227-233.
[21] 邓家华,吕其壮,陈艳,等.猪圆环病毒3型分子生物学研究进展[J].畜牧与兽医,2019,51(9):125-128. DENG J H,LYU Q Z,CHEN Y,et al.Advances in molecular biology of porcine circovirus type 3[J].Animal Husbandry and Veterinary Medicine,2019,51(9):125-128.(in Chinese)
[22] 张献.核定位信号对病毒衣壳蛋白在E.coli中表达的影响及重组衣壳蛋白免疫学活性研究[D].长春:吉林大学,2019. ZHANG X.Effect of nuclear localization signal on expression of viral capsid protein in E.coli and immunological activity of recombinant capsid protein[D].Changchun:Jilin University,2019.(in Chinese)
[23] 连凯琪,周玲玲,张明亮,等.新发猪圆环病毒3型Cap蛋白的原核表达和纯化[J].河南农业科学,2018,47(11):120-123. LIAN K Q,ZHOU L L,ZHANG M L,et al.Prokaryotic expression and purification of Cap protein of new porcine circovirus type 3[J].Journal of Henan Agricultural Sciences,2008,47(11):120-123.(in Chinese)
[24] 陈桂华,孙媛,周玲,等.猪圆环病毒3型Cap蛋白的表达纯化及单克隆抗体的制备[J].中国兽医科学,2019,49(1):13-19. CHEN G H,SUN Y,ZHOU L,et al.Expression and purification of Cap protein of porcine circovirus type 3 and preparation of monoclonal antibody[J].Chinese Veterinary Science,2019,49(1):13-19.(in Chinese)
[25] 郭奎,徐朋丽,赵宇,等.PCV3河南株Cap基因的克隆、测序及其在大肠杆菌中的表达[J].中国兽医学报,2018,38(11):2063-2067. GUO K,XU P L,ZHAO Y,et al.Cloning,sequencing and expression of Cap gene of Henan strain PCV3 in Escherichia coli[J].Chinese Journal of Veterinary Science,2008,38(11):2063-2067.(in Chinese)
[26] 王帅勇,汪琪,朱世强,等.猪圆环病毒3型Cap蛋白的原核表达[J].中国动物传染病学报,2019,9:1-8. WANG S Y,WANG Q,ZHU S Q,et al.Prokaryotic expression of Cap protein of porcine circovirus type 3[J].Chinese Journal of Animal Infectious Diseases,2019,9:1-8.(in Chinese)

Prokaryotic Expression and Purification of Cap Protein of Xinjiang Porcine Circovirus Type 3 Strain

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	JIAO Linlin, CHENG Yuting, WU Qingguo, WU Shuang, WU Zhi, ZHU Shanyuan, QIAN Yingjuan. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Tembusu Virus Capsid Protein [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2395-2402.
[2]	LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.
[3]	SONG Yue, SU Shengjie, ZHANG Fan, BAI Fan, DAI Lingli, WANG Na, WANG Dawei, YANG Qianwen, ZHAO Shihua, ZHANG Yuemei. Prokaryotic Expression and Polyclonal Antibody Preparation of Mannheimia haemolytica Truncated Leukotoxin [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2479-2486.
[4]	ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.
[5]	HU Leyu, HU Xinyao, LI Ying, ZHAO Shengmiao, SHEN Fengzhen, WANG Changfa, LI Liangliang, WANG Tongtong, REN Huiying. Bioinformatics Analysis of HO-1 Gene and Preparation of Polyclonal Antibody in Dezhou Donkey [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1499-1510.
[6]	WANG Wenjie, RU Penghui, YU Chuan, DU Fuxi, CHEN Songbiao, SHANG Ke, ZHANG Chunjie, CHENG Xiangchao. Prokaryotic Expression of VP2 Protein of Luoyang Feline Panleukopenia Virus Isolate and Polyclonal Antibody Preparation [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1511-1521.
[7]	LIU Jiaxing, HAN Xueying, ZHANG Xinru, DENG Jiaxin, YAO Lunguang, LIU Yangkun. Preparation and Immunogenicity Analysis of Ferritin Nanoparticles Carrying Antigenic Epitopes of Porcine Epidemic Diarrhea Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1567-1574.
[8]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[9]	ZHANG Zhitao, LI Xianglong, KONG Lingli, LUO Yuxin, WANG Dongying. Prokaryotic Expression of Serine/Threonine Protein Phosphatase 2A in Fasciola gigantica and Expression Analysis at Different Stages [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1107-1117.
[10]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[11]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[12]	ZHANG Kai, ZHANG Chunping, GE Shengqiang, SUN Chunxi, CHENG Jie, FU Chunyu, WANG Gang, NIU Xing, PENG Jun. Expression of p22 Protein of African Swine Fever Virus,Preparation of Polyclonal Antibody and Establishment of Indirect ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 270-279.
[13]	ZHU Lilin, WANG Houkun, CHEN Xueyang, LIU Jing, LIANG Xiongyan, FANG Chun, YANG Yuying. Effect of Chicken IFIT5 on Replication of J Subgroup Avian Leukemia Virus Within DF-1 Cells [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 310-316.
[14]	WANG Yuchen, TIAN Guangyuan, ZHOU Yaping, GUO Ting, ZHAO Hongmei, SUN Yajie, ZHAO Fengmiao, BIAN Yuchen, YU Jialiang, HAO Yongqing. Prokaryotic Expression of BPIV3 NP Protein and Its Immunoenhancing Effect on the BPIV3 Inactivated Vaccine [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3122-3130.
[15]	WANG Wei, HAN Huihui, DING Naizheng, HE Chengqiang. Prokaryotic Expression of Bovine Viral Diarrhea Virus E0 Protein and Preparation of Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(8): 3131-3139.