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
|
摘要
试验旨在表达牛乳头瘤病毒(BPV)的L1衣壳蛋白,并制备其多克隆抗体。对疑似牛乳头状瘤的病料进行病理组织学检查,采用L1基因简并引物FAP59/FAP64进行扩增,然后设计特异性引物,将衣壳蛋白L1基因亚克隆至pET-30a(+)表达载体中,构建重组原核表达载体pET30a-BPV2-L1,在E.coli Rosetta~(TM)(DE3) pLysS宿主菌中表达重组蛋白rBPV2-L1,并以纯化的rBPV2-L1蛋白为免疫原制备兔抗BPV2-L1蛋白多克隆抗体,随后间接ELISA检测其效价,并进行间接免疫荧光试验分析。病理组织切片观察结果显示,病变部分表皮细胞增生、角质过度并出现细胞挖空等BPV感染的组织病变情况;序列分析确定BPV分离株的基因型为2型,L1基因编码区长1 494 bp,可编码一个含有497个氨基酸的衣壳蛋白,蛋白分子质量为55.5 ku;与各型BPV的L1氨基酸序列系统进化树分析结果显示,其与BPV2-SW01(GenBank登录号:KC878306.1)、BPV2(GenBank登录号:KX113620.1)处于同一遗传进化分支,且属于Delta属成员;诱导表达的rBPV2-L1蛋白主要以包涵体形式存在于沉淀中;间接ELISA法检测多克隆抗体效价高达1∶163 840,间接免疫荧光分析表明,兔抗BPV2-L1多克隆抗体能与瞬时表达的BPV2-L1蛋白发生特异性反应。以上结果证实,本研究成功克隆、表达了BPV2 L1基因,且重组蛋白rBPV2-L1具有较好的免疫原性,所制备的BPV2-L1蛋白多克隆抗体具有良好的免疫活性,为进一步研究BPV2亚单位疫苗奠定基础。
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-30 a(+) expression vector to construct recombinant prokaryotic expression vector pET30 a-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.
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-30 a(+) expression vector to construct recombinant prokaryotic expression vector pET30 a-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.
引文
[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 4 L1 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.
[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 4 L1 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.