牛乳头瘤病毒2型L1基因的原核表达及多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2019.06.029

Abstract

Abstract:

The test was aimed to express the L1 capsid protein of bovine papillomavirus (BPV),and prepare its polyclonal antibodies.Histopathological examination was conducted using the cutaneous papilloma samples of cattle.The L1 gene degenerate primer FAP59/FAP64 was used for amplification,and then specific primers were designed.The capsid protein L1 gene was subcloned into pET-30a(+) expression vector to construct recombinant prokaryotic expression vector pET30a-BPV2-L1.The recombinant protein rBPV2-L1 was expressed in E.coli Rosetta^TM(DE3)pLysS host strain,and the rabbit anti-BPV2-L1 protein polyclonal antibody was prepared by using purified rBPV2-L1 protein as immunogen,and then the titer was detected by indirect ELISA,indirect immunofluorescence assay was performed.The pathological tissue section observation showed that the epidermal cells proliferated,the keratin was excessive,and the tissue lesions of BPV infection such as cell hollowing occurred.Sequence analysis confirmed that the genotype of the BPV isolated strain was type 2,and the coding region of L1 gene was 1 494 bp,which could encode a capsid protein containing 497 amino acids with a molecular mass of 55.5 ku.Phylogenetic tree analysis with L1 amino acid sequence of each type of BPV showed that it was in the same genetic evolution branch as BPV2-SW01 (GenBank accession No.:KC878306.1) and BPV2 (GenBank accession No.:KX113620.1),and belonged to the genus Delta.The induced expression of rBPV2-L1 protein was mainly present in the precipitate in the form of inclusion bodies.The indirect ELISA assay results showed that the polyclonal antibody titer was as high as 1:163 840.Indirect immunofluorescence analysis results showed that the rabbit anti-BPV2-L1 polyclonal antibody reacted specifically with the transiently expressed BPV2-L1 protein.The above results confirmed that the BPV2 L1 gene was successfully cloned and expressed in this study,and the recombinant protein rBPV2-L1 had good immunogenicity.The prepared polyclonal antibody against BPV2-L1 protein had good immunological activity,and laid the foundation for further study of the BPV2 subunit vaccine.

Key words: bovine papilloma virus(BPV); L1 gene; prokaryotic expression; polyclonal antibody

CLC Number:

S852.65⁺3

DU Jiwen, LIU Yue, SONG Zhifeng, LU Zhaoxiang, XU Weicheng, LI Mingzhu, GAO Mingchun, WANG Junwei. Prokaryotic Expression of Bovine Papillomavirus Type 2 L1 Gene and Preparation of Polyclonal Antibody[J]. , 2019, 46(6): 1808-1815.

References

[1] ROPERTO S,RUSSO V,CORRADO F,et al.Oral fibropapillomatosis and epidermal hyperplasia of the lip in newborn lambs associated with bovine Deltapapillomavirus[J].Scientific Reports,2018,8(1):13310.
[2] ARALDI R P,GIOVANNI D N,MELO T C,et al.Bovine papillomavirus isolation by ultracentrifugation[J].Journal of Virological Methods,2014,208:119-124.
[3] BAM J,KUMAR P,LEISHANGTHEM G D,et al.Spontaneous cutaneous papillomatosis in yaks and detection and quantification of bovine papillomavirus-1 and -2[J].Transboundary and Emerging Diseases,2013,60(5):475-480.
[4] OGAWA T,TOMITA Y,OKADA M,et al.Broad-spectrum detection of papillomaviruses in bovine teat papillomas and healthy teat skin[J]. Journal of General Virology,2004,85(Pt 8):2191-2197.
[5] ARALDI R P,MELO T C,DINIZ N,et al.Bovine papillomavirus clastogenic effect analyzed in comet assay[J].BioMed Research International,2013,2013:630683.
[6] BERNARD H U,BURK R D,CHEN Z,et al.Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments[J].Virology,2010,401(1):70-79.
[7] LUNARDI M,DE ALCANTARA B K,OTONEL R A,et al.Bovine papillomavirus type 13 DNA in equine sarcoids[J].Journal of Clinical Microbiology,2013,51(7):2167-2171.
[8] 贺志昊.新疆北疆地区牛乳头瘤病毒流行株基因分型及其衣壳蛋白表达研究[D].石河子:石河子大学,2014. HE Z H.Genotyping and expression of capsid protein of bovine papillomavirus in Northern Xinjiang[D].Shihezi:Shihezi University,2014.(in Chinese)
[9] 王青青,石长青,胡建军,等.新疆南疆牛乳头状瘤病毒鉴定与基因分型[J].畜牧与兽医,2015,47(11):99-103. WANG Q Q,SHI C Q,HU J J,et al.Idetificationand genotyping of bovine papillomavirus in Southern Xinjiang[J].Animal Husbandry & Veterinary Medicine,2015,47(11):99-103.(in Chinese).
[10] 张海,张军,杨忠成,等.牛乳头瘤病毒基因2型贵州GZ01流行株全基因组序列测定及其特征分析[J].基因组学与应用生物学,2017,3(12):5090-5098. ZHANG H,ZHANG J,YANG Z C,et al.Sequence analysis of the complete genome of bovine papillomavirus type 2 genotype GZ01 strain from skin samples of cows in Guizhou[J].Genomics and Applied Biology,2017,3(12):5090-5098.(in Chinese).
[11] 张海,周碧君,杨忠成,等.牛乳头状瘤病毒贵州株L1基因克隆及生物信息学分析[J].中国畜牧兽医,2016,43(11):2834-2843. ZHANG H,ZHOU B J,YANG Z C,et al.Cloning and bioinformatic analysis of L1 gene of BPV form Guizhou provine[J].China Animal Husbandry & Veterinary Medicine,2016,43(11):2834-2843.(in Chinese).
[12] GILLISON M L,ALEMANY L,SNIJDERS P J,et al.Human papillomavirus and diseases of the upper airway:Head and neck cancer and respiratory papillomatosis[J].Vaccine,2012,30(Suppl 5):F34-F54.
[13] YANG A,PENG S,FARMER E,et al.Enhancing antitumor immunogenicity of HPV16-E7 DNA vaccine by fusing DNA encoding E7-antigenic peptide to DNA encoding capsid protein L1 of Bovine papillomavirus[J].Cell & Bioscience,2017,7:46.
[14] BRANDT S,APPRICH V,HACKL V,et al.Prevalence of bovine papillomavirus and Treponema DNA in bovine digital dermatitis lesions[J].Veterinary Microbiology,2011,148(2-4):161-167.
[15] ARALDI R P,ASSAF S,CARVALHO R F,et al.Papillomaviruses:A systematic review[J].Genetics and Molecular Biology,2017,40(1):1-21.
[16] MAEDA Y,SHIBAHARA T,WADA Y,et al.An outbreak of teat papillomatosis in cattle caused by bovine papilloma virus (BPV) type 6 and unclassified BPVs[J].Veterinary Microbiology,2007,121(3-4):242-248.
[17] ROPERTO S,MUNDAY J S,CORRADO F,et al.Detection of bovine papillomavirus type 14 DNA sequences in urinary bladder tumors in cattle[J].Veterinary Microbiology,2016,190:1-4.
[18] MAZZUCHELLI-DE-SOUZA J,DE CARVALHO R F,MODOLO D G,et al.First detection of bovine papillomavirus type 2 in cutaneous wart lesions from ovines[J].Transboundary and Emerging Diseases,2018,65(4):939-943.
[19] KOCH C,RAMSAUER A S,DROGEMULLER M,et al.Genomic comparison of bovine papillomavirus 1 isolates from bovine,equine and asinine lesional tissue samples[J].Virus Research, 2018,244:6-12.
[20] 易驰喆,孙文超,郑敏,等.牛乳头瘤病毒基因1型广西GX01流行株全基因组克隆及序列分析[J].中国畜牧兽医,2018,45(7):1731-1739. YI C Z,SUN W C,ZHENG M,et al.Sequence analysis of the complete genome of bovine papillomavirus type 1 genotype GX01 strain from skin samples of cows in Guangxi[J]. China Animal Husbandry & Veterinary Medicine,2018,45(7):1731-1739.(in Chinese).
[21] LIMA E G,LIRA R C,JESUS A L,et al.Development of a DNA-based vaccine strategy against bovine papillomavirus infection,involving the E5 or L2 gene[J].Genetics and Molecular Research,2014,13(1):1121-1126.
[22] RIBEIRO-MULLER L,MULLER M.Prophylactic papillomavirus vaccines[J].Clinics in Dermatology,2014,32(2):235-247.
[23] HAINISCH E K,ABEL-REICHWALD H,SHAFTI-KERAMAT S,et al.Potential of a BPV1 L1 VLP vaccine to prevent BPV1-or BPV2-induced pseudo-sarcoid formation and safety and immunogenicity of EcPV2 L1 VLPs in horse[J].The Journal of General Virology,2017,98(2):230-241.
[24] WATANABE S,ⅡZUKA T,HATAMA S,et al.Production of highly immunogenic virus-like particles of bovine papillomavirus type 6 in silkworm pupae[J].Vaccine,2017,35(43):5878-5882.
[25] JESUS A L,MARIZ F C,SOUZA H M,et al.Expression of the bovine papillomavirus type 1,2 and 4L1 genes in the yeast Pichia pastoris[J].Genetics and Molecular Research,2012,11(3):2598-2607.
[26] MODOLO D G,ARALDI R P,MAZZUCHELLI-DESOUZA J,et al.Integrated analysis of recombinant BPV-1 L1 protein for the production of a bovine papillomavirus VLP vaccine[J].Vaccine,2017,35(12):1590-1593.

Prokaryotic Expression of Bovine Papillomavirus Type 2 L1 Gene and Preparation of Polyclonal Antibody

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]	OU Yunwen, PAN Qin, WANG Yang, DAI Junfei, REN Shaoke, ZHANG Yang, ZHANG Jie. Truncated Expression of ORF2 Gene of Porcine Parvovirus Type 6 and Preparation of Its Polyclonal Antibody [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2487-2495.
[5]	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.
[6]	WANG Xueping, ZHANG Xiaojun, LI Xiaoyue, WANG Guodong, ZHANG Fuliang, SONG Yuwei, ZHANG Mingliang, MA Lei. Prokaryotic Expression of Hexon Protein of Highly Pathogenic Fowl Adenovirus Serotype 4 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2053-2060.
[7]	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.
[8]	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.
[9]	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.
[10]	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.
[11]	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.
[12]	NIU Xiaojie, XU Mingguo, XIAO Li, LIU Wenhao, CHEN Chuangfu, WANG Yong. Expression and Purification of IPAJ Protein of Salmonella Pullorum, Preparation of Polyclonal Antibody and Establishment of ELISA Method [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1218-1228.
[13]	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.
[14]	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.
[15]	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.