BPV1贵州株L1基因序列分析及原核表达
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摘要
为表达牛乳头瘤病毒1型贵州株(BPV1-GZLZ株)衣壳蛋白L1基因,采用PCR方法从贵州省六枝特区某规模化养牛场疑似病牛的皮肤肿瘤病料样品中扩增BPV1 L1基因,克隆至p MD19-T载体,测序后进行生物信息学分析;同时将BPV1-GZLZ株L1基因克隆于原核表达质粒p ColdⅠ中,经IPTG诱导L1蛋白表达,并对重组蛋白进行SDS-PAGE和Western blotting分析。生物信息学分析显示,BPV1-GZLZ株L1基因核苷酸序列长为1488 bp,编码495个氨基酸,分子结构式为C2484H3884N678O737S16,理论等电点(PI)为8.68,二级结构主要包括α-螺旋、无规则卷曲和β-折叠为主,无跨膜结构域和信号肽; BPV1-GZLZ L1基因序列与BPV1参考株的核苷酸和氨基酸同源性均为99.8%,处在同一遗传进化分支,且与BPV13和BPV2、BPV2-GZ01的亲缘关系较近; Western blotting结果显示,经IPTG诱导的重组表达蛋白能与His单抗产生特异性反应,条带为55.6 kD左右与SDS-PAGE结果一致。研究表明,试验构建了BPV1-GZLZ株L1基因原核表达载体并成功表达。
In order to express the capsid protein L1 gene of bovine papillomavirus type 1 Guizhou strain( BPV1-GZLZ strain). The BPV1 L1 gene was amplified from the skin tumor samples of suspected diseased cattle in a large-scale cattle farm in Liuzhi District,Guizhou Province by PCR,cloned into pMD19-T vector,and sequenced for bioinformatics analysis. Then L1 gene of BPV1-GZLZ strain was cloned into prokaryotic expression plasmid pCold I,L1 protein expression was induced by IPTG,and SDS-PAGE and Western blotting analysis were performed on recombinant protein. Bioinformatics analysis showed that the nucleotide sequence of L1 gene of BPV1-GZLZ strain was 1488 bp,encoding 495 amino acids,the molecular structure was C2484H3884N678O737S16,the theoretical isoelectric point( PI) was 8.68,and the secondary structure mainly consisted of α-helix. Irregular coiling and β-sheet-based,no transmembrane domain and signal peptide; the nucleotide and amino acid homology of BPV1-GZLZ L1 gene sequence and BPV1 reference strain were both 99.8%,in the same genetic evolution branch And the relationship with BPV13 and BPV2,BPV2-GZ01 were closer; Western blotting results showed that the recombinantly expressed protein induced by IPTG could specifically react with His monoclonal antibody,and the band was about 55.6 k Da consistent with SDS-PAGE results. The prokaryotic expression vector of L1 gene of BPV1-GZLZ strain was constructed and successfully expressed in this study.
In order to express the capsid protein L1 gene of bovine papillomavirus type 1 Guizhou strain( BPV1-GZLZ strain). The BPV1 L1 gene was amplified from the skin tumor samples of suspected diseased cattle in a large-scale cattle farm in Liuzhi District,Guizhou Province by PCR,cloned into pMD19-T vector,and sequenced for bioinformatics analysis. Then L1 gene of BPV1-GZLZ strain was cloned into prokaryotic expression plasmid pCold I,L1 protein expression was induced by IPTG,and SDS-PAGE and Western blotting analysis were performed on recombinant protein. Bioinformatics analysis showed that the nucleotide sequence of L1 gene of BPV1-GZLZ strain was 1488 bp,encoding 495 amino acids,the molecular structure was C2484H3884N678O737S16,the theoretical isoelectric point( PI) was 8.68,and the secondary structure mainly consisted of α-helix. Irregular coiling and β-sheet-based,no transmembrane domain and signal peptide; the nucleotide and amino acid homology of BPV1-GZLZ L1 gene sequence and BPV1 reference strain were both 99.8%,in the same genetic evolution branch And the relationship with BPV13 and BPV2,BPV2-GZ01 were closer; Western blotting results showed that the recombinantly expressed protein induced by IPTG could specifically react with His monoclonal antibody,and the band was about 55.6 k Da consistent with SDS-PAGE results. The prokaryotic expression vector of L1 gene of BPV1-GZLZ strain was constructed and successfully expressed in this study.
引文
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[2]Ataseven V S,zgür Kanat,Ergün Y.Molecular identification of bovine papillomaviruses in dairy and beef cattle:First description of Xi-and Epsilon papillomaviruses in Turkey[J].Turkish Journal of Veterinary&Animal Sciences,2016,40(6):757-763.
[3]Leal A M,Ferraz O P,Carvalho C,et al.Quercetin induces structural chromosomal aberrations and uncommon rearrangements in bovine cells transformed by the E7 protein of bovine papillomavirus type 4[J].Veterinary&Comparative Oncology,2010,1(1):15-21.
[4]Borzacchiello G,Roperto F.Bovine papillomaviruses,papillomas and cancer in cattle[J].Veterinary Research,2008,39(5):45.
[5]Modis Y,Trus B L,Harrison S C.Atomic model of the papillomavirus capsid[J].Embo Journal,2014,21(18):4754-4762.
[6]Campo M S.Papillomavirus and disease in humans and animals.[J].Veterinary&Comparative Oncology,2010,1(1):3-14.
[7]Pang F,Shi Q,Du L,et al.Complete genome sequence of bovine papillomavirus genotype 13 from local yellow cattle in Hainan province,China.[J].Genome Announcements,2014,2(6).doi:10.1128/genome A.01087-14.
[8]Doorbar J.The papillomavirus life cycle[J].Journal of Clinical Virology,2005,32(1 Suppl):7-15.
[9]Freitas A C,Silva,M.A R,et al.Recent insights into Bovine Papillomavirus[J].African Journal of Microbiology Research,2011,5(33):6004-6012.
[10]Lowy D R,Frazer I H.Chapter 16:Prophylactic human papillomavirus vaccines[J].J Natl Cancer Inst Monogr,2003,2003(31):111-116.
[11]McGrath M,de Villiers G K,Shephard E,et al.Development of human papillomavirus chimaeric L1/L2 candidate vaccines[J].Archives of Virology,2013,159(10):2079-2088.
[12]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.
[13]梁辰,姜晓文,刘旭,等.牛乳头状瘤的病理学观察及其病毒基因型分析[J].中国预防兽医学报,2013,35(5):423-425.Liang C,Jiang X W,Liu X,et al.The pathologic examination of bovine papilloma and the virus genetype analysis[J].Chinese Journal of Preventive Veterinary Medicine,2013,35(5):423-425.
[14]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&Molecular Research Gmr,2014,13(1):1121-1126.
[15]彭昊,吴翠兰,李军,等.广西黄牛乳头状瘤病毒的鉴定及其基因组序列分析[J].南方农业学报,2018,49(3):563-571.Peng H,Wu C L,Li J,et al.Identification of bovine papillomavirus and genome sequence analysis for cattle in Guangxi[J].Journal of Southern Agriculture,2018,49(3):563-571.