羊口疮病毒安徽株023基因的原核表达与生物信息学分析
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摘要
试验旨在克隆表达羊口疮病毒安徽株023基因,对该基因进行PCR扩增、亚克隆及表达,通过蛋白表达纯化后进行SDS-PAGE和Western blot鉴定,并利用生物信息学软件预测该基因编码的蛋白的理化性质、二级结构、信号肽、跨膜结构域等。结果显示,023基因全长819 bp,编码272个氨基酸,蛋白大小约为42 ku,主要以包涵体的形式表达且有良好的反应原性。生物信息学分析表明,该蛋白是亲水稳定蛋白,其中α-螺旋(h)占27. 57%,延伸链(e)占17. 28%,β-转角(t)占12. 13%,无规则卷曲(c)占43. 01%,无信号肽区域和跨膜结构域,可能存在30个磷酸化位点,4个B细胞抗原表位,3个CTL表位和3个Th表位。研究结果不仅有助于了解羊口疮病毒023基因的结构与功能,还能为建立快速诊断的检测方法提供参考。
Our study was aimed at cloning and expressing the 023 gene of orf virus (ORFV) Anhui strain,and predicting its biological characteristics. The 023 gene was amplified by PCR,then successfully expressed using a prokaryotic vector. Then,the expressed protein was purified,and identified by SDS-PAGE and Western-blot. In addition,the physicochemical properties,secondary structure,signal peptide,transmembrane domain of the protein was analyzed by bioinformatics software. The results showed that the 023 gene of ORFV Anhui strain was 819 bp in length which could encode 272 amino acids. The size of expressed protein was approximately 42 ku,which was mainly expressed in inclusion body form with good reactogenicity. The results from bioinformatics analysis showed that the protein was a hydrophilic stable protein. There were 27. 57% α-helix (h),17. 28% extended chain (e),12. 13%β-turn (t),43. 01% random coli (c). There was no signal peptide region and transmembrane domain. Maybe,it had 30 phosphorylation sites. Moreover,there were four B cell epitopes,three CTL epitopes and three Th epitopes.In conclusion,our study could provide a reference for further study of the structure and function of ORFV 023 gene.
Our study was aimed at cloning and expressing the 023 gene of orf virus (ORFV) Anhui strain,and predicting its biological characteristics. The 023 gene was amplified by PCR,then successfully expressed using a prokaryotic vector. Then,the expressed protein was purified,and identified by SDS-PAGE and Western-blot. In addition,the physicochemical properties,secondary structure,signal peptide,transmembrane domain of the protein was analyzed by bioinformatics software. The results showed that the 023 gene of ORFV Anhui strain was 819 bp in length which could encode 272 amino acids. The size of expressed protein was approximately 42 ku,which was mainly expressed in inclusion body form with good reactogenicity. The results from bioinformatics analysis showed that the protein was a hydrophilic stable protein. There were 27. 57% α-helix (h),17. 28% extended chain (e),12. 13%β-turn (t),43. 01% random coli (c). There was no signal peptide region and transmembrane domain. Maybe,it had 30 phosphorylation sites. Moreover,there were four B cell epitopes,three CTL epitopes and three Th epitopes.In conclusion,our study could provide a reference for further study of the structure and function of ORFV 023 gene.
引文
[1]SPYROU V,VALIAKOS G.Orf virus infection in sheep or goats[J].Veterinary Microbiology,2015,181(1/2):178-182.
[2]KUMAR N,WADHWA A,CHAUBEY K K,et al.Isolation and phylogenetic analysis of an orf virus from sheep in Makhdoom,India[J].Virus Genes,2014,48(2):312-319.
[3]ZHANG K,LIU Y,KONG H,et al.Comparison and phylogenetic analysis based on the B2L gene of orf virus from goats and sheep in China during 2009-2011[J].Archives of Virology,2014,159(6):1475-1479.
[4]Al-QATTAN M M.Orf infection of the hand[J].Journal of Hand Surgery,2011,36(11):1855-1858.
[5]FLEMING S B,WISE L M,MERCER A A.Molecular genetic analysis of orf virus:a poxvirus that has adapted to skin[J].Viruses,2015,7(3):1505-1539.
[6]王勇,赵玉洁,杨侃侃,等.口疮病毒020基因的克隆、表达及多抗制备[J].安徽农业大学学报,2017,44(5):780-783.WANG Y,ZHAO Y J,YANG K K,et al.Cloning and expression of the Orf virus 020 gene and its polyclonal antibody preparation[J].Journal of Anhui Agricultural University,2017,44(5):780-783.(in Chinese with English abstract)
[7]闫丰超,邵佳,窦永喜.羊口疮病毒分子生物学的研究进展[J].中国兽医科学,2013(1):103-109.YAN F C,SHAO J,DOU Y X.Progress in molecular biology of orf virus[J].Chinese Veterinary Science,2013(1):103-109.(in Chinese with English abstract)
[8]葛士坤,张凯照,鲁荣,等.2006-2016年中国羊口疮病毒的遗传演化分析[J].中国畜牧兽医,2018,45(2):439-447.GE S K,ZHANG K Z,LU R,et al.Genetic and phylogenetic analysis of orf virus isolated in China from 2006 to 2016[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):439-447.(in Chinese with English abstract)
[9]SPYROU V,VALIAKOS G.Orf virus,infection in sheep or goats[J].Veterinary Microbiology,2015,181(1/2):178-182.
[10]成伟伟,张克山,刘永杰,等.羊传染性脓疱病毒基因组结构和主要基因功能研究进展[J].动物医学进展,2014,35(7):82-85.CHENG W W,ZHANG K S,LIU Y J,et al.Advance in genome structure and major gene function of orf virus[J].Progress in Veterinary Medicine,2014,35(7):82-85.(in Chinese with English abstract)
[11]FRIEBE A,FRIEDERICHS S,SCHOLZ K,et al.Characterization of immunostimulatory components of orf virus(parapoxvirus ovis)[J].Journal of General Virology,2011,92(Pt 7):1571-1584.
[12]WANG Y,YANG K,BAI C,et al.Development of a SYBRGreenⅠreal-time PCR for the detection of the orf virus[J].AMB Express,2017,7(1):21.
[13]YU Y,TAN Q,ZHAO W,et al.Characterization of an orf virus isolate from an outbreak in Heilongjiang province,China[J].Archives of Virology,2017,162(10):1-7.
[14]CHEN H,LI W,KUANG Z,et al.The whole genomic analysis of orf virus strain HN3/12 isolated from Henan province,central China[J].BMC Veterinary Research,2017,13(1):260.
[15]CHI X,ZENG X,LUO S.Diagnosis and phylogenetic analysis of a multifocal cutaneous orf virus with mixed bacterial infection outbreak in goats in Fujian province,China[J].Archives of Virology,2017(10):1-10.
[16]王光祥,尚佑军,吕占禄,等.羊口疮病毒B2L蛋白二级结构及其细胞表位的预测[J].中国兽医科学,2012,42(11):1133-1138.WANG G X,SHANG Y J,LYU Z L,et al.Prediction of secondary structure and cell epitopes of B2L protein of orf virus[J].Chinese Veterinary Science,2012,42(11):1133-1138.(in Chinese with English abstract)
[17]黄宇良,汪立平,陆克文,等.片球菌素ped A基因的原核表达、纯化及生物信息学分析[J].生物技术通报,2017,33(12):144-150.HUANG Y L,WANG L P,LU K W,et al.Prokaryotic expression,purification and bioinformatics analysis of pediocin pedA gene[J].Biotechnology Bulletin,2017,33(12):144-150.(in Chinese with English abstract)
[18]BHASIN M,RAGHAVA G P.Prediction of CTL epitopes using QM,SVM and ANN techniques[J].Vaccine,2004,22(23/24):3195-3204.
[2]KUMAR N,WADHWA A,CHAUBEY K K,et al.Isolation and phylogenetic analysis of an orf virus from sheep in Makhdoom,India[J].Virus Genes,2014,48(2):312-319.
[3]ZHANG K,LIU Y,KONG H,et al.Comparison and phylogenetic analysis based on the B2L gene of orf virus from goats and sheep in China during 2009-2011[J].Archives of Virology,2014,159(6):1475-1479.
[4]Al-QATTAN M M.Orf infection of the hand[J].Journal of Hand Surgery,2011,36(11):1855-1858.
[5]FLEMING S B,WISE L M,MERCER A A.Molecular genetic analysis of orf virus:a poxvirus that has adapted to skin[J].Viruses,2015,7(3):1505-1539.
[6]王勇,赵玉洁,杨侃侃,等.口疮病毒020基因的克隆、表达及多抗制备[J].安徽农业大学学报,2017,44(5):780-783.WANG Y,ZHAO Y J,YANG K K,et al.Cloning and expression of the Orf virus 020 gene and its polyclonal antibody preparation[J].Journal of Anhui Agricultural University,2017,44(5):780-783.(in Chinese with English abstract)
[7]闫丰超,邵佳,窦永喜.羊口疮病毒分子生物学的研究进展[J].中国兽医科学,2013(1):103-109.YAN F C,SHAO J,DOU Y X.Progress in molecular biology of orf virus[J].Chinese Veterinary Science,2013(1):103-109.(in Chinese with English abstract)
[8]葛士坤,张凯照,鲁荣,等.2006-2016年中国羊口疮病毒的遗传演化分析[J].中国畜牧兽医,2018,45(2):439-447.GE S K,ZHANG K Z,LU R,et al.Genetic and phylogenetic analysis of orf virus isolated in China from 2006 to 2016[J].China Animal Husbandry&Veterinary Medicine,2018,45(2):439-447.(in Chinese with English abstract)
[9]SPYROU V,VALIAKOS G.Orf virus,infection in sheep or goats[J].Veterinary Microbiology,2015,181(1/2):178-182.
[10]成伟伟,张克山,刘永杰,等.羊传染性脓疱病毒基因组结构和主要基因功能研究进展[J].动物医学进展,2014,35(7):82-85.CHENG W W,ZHANG K S,LIU Y J,et al.Advance in genome structure and major gene function of orf virus[J].Progress in Veterinary Medicine,2014,35(7):82-85.(in Chinese with English abstract)
[11]FRIEBE A,FRIEDERICHS S,SCHOLZ K,et al.Characterization of immunostimulatory components of orf virus(parapoxvirus ovis)[J].Journal of General Virology,2011,92(Pt 7):1571-1584.
[12]WANG Y,YANG K,BAI C,et al.Development of a SYBRGreenⅠreal-time PCR for the detection of the orf virus[J].AMB Express,2017,7(1):21.
[13]YU Y,TAN Q,ZHAO W,et al.Characterization of an orf virus isolate from an outbreak in Heilongjiang province,China[J].Archives of Virology,2017,162(10):1-7.
[14]CHEN H,LI W,KUANG Z,et al.The whole genomic analysis of orf virus strain HN3/12 isolated from Henan province,central China[J].BMC Veterinary Research,2017,13(1):260.
[15]CHI X,ZENG X,LUO S.Diagnosis and phylogenetic analysis of a multifocal cutaneous orf virus with mixed bacterial infection outbreak in goats in Fujian province,China[J].Archives of Virology,2017(10):1-10.
[16]王光祥,尚佑军,吕占禄,等.羊口疮病毒B2L蛋白二级结构及其细胞表位的预测[J].中国兽医科学,2012,42(11):1133-1138.WANG G X,SHANG Y J,LYU Z L,et al.Prediction of secondary structure and cell epitopes of B2L protein of orf virus[J].Chinese Veterinary Science,2012,42(11):1133-1138.(in Chinese with English abstract)
[17]黄宇良,汪立平,陆克文,等.片球菌素ped A基因的原核表达、纯化及生物信息学分析[J].生物技术通报,2017,33(12):144-150.HUANG Y L,WANG L P,LU K W,et al.Prokaryotic expression,purification and bioinformatics analysis of pediocin pedA gene[J].Biotechnology Bulletin,2017,33(12):144-150.(in Chinese with English abstract)
[18]BHASIN M,RAGHAVA G P.Prediction of CTL epitopes using QM,SVM and ANN techniques[J].Vaccine,2004,22(23/24):3195-3204.