斯氏副柔线虫SHR基因表达载体的构建及生物信息学分析
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摘要
为探讨斯氏副柔线虫免疫相关SHR基因作为疫苗候选抗原及早期诊断抗原的可能性,提取斯氏副柔线虫的总RNA,用RT-PCR技术扩增SHR基因,并将PCR产物克隆到pMD19-T载体,构建原核表达载体pET-30a(+)-SHR,利用在线软件对该基因编码蛋白序列进行生物信息学分析。结果表明:克隆获得SHR基因全长1 083 bp,编码360个氨基酸,理论等电点为6.09,无信号肽和跨膜区;磷酸化预测含有29个磷酸化位点;结构分析发现,α-螺旋构成二级结构的主要成分,亲水性氨基酸比例超过60%;抗原表位预测表明,SHR蛋白是一种抗原性较高的亲水性蛋白。推测SHR有望用作斯氏副柔线虫的免疫诊断抗原和疫苗候选抗原。
The possibility of SHR gene as a vaccine candidate antigen and early diagnostic antigen was preliminary discussed in this research.The total RNA was extracted from Parabronema skrjabini and SHR gene was cloned by RT-PCR.The PCR product was cloned into pMD19-T and a prokaryotic expression vector pET-30 a(+)-SHR was then constructed.The protein structure and antigenic epitopes of SHR gene were analyzed by using online bioinformatics software.The results indicated that SHR gene was 1 083 bp in length,encoding 360 aminoacids and its theoretical pI is 6.09.Transmembrane structure and signal peptide analysis showed that the SHR protein contained a type of hydrophilic no transmembrane domains and signal peptide region and 29 phosphorylation sites.The secondary structure of SHR was mainly composed of alpha helix and the proportion of hydrophilic amino was over 60%.The antigenic epitopes prediction results indicated that SHR protein was a hydrophilic protein with high antigenicity.These results suggested that the SHR antigen has strong potential to be a candidate for immune diagnostics and used as a vaccine against P.skrjabini.
The possibility of SHR gene as a vaccine candidate antigen and early diagnostic antigen was preliminary discussed in this research.The total RNA was extracted from Parabronema skrjabini and SHR gene was cloned by RT-PCR.The PCR product was cloned into pMD19-T and a prokaryotic expression vector pET-30 a(+)-SHR was then constructed.The protein structure and antigenic epitopes of SHR gene were analyzed by using online bioinformatics software.The results indicated that SHR gene was 1 083 bp in length,encoding 360 aminoacids and its theoretical pI is 6.09.Transmembrane structure and signal peptide analysis showed that the SHR protein contained a type of hydrophilic no transmembrane domains and signal peptide region and 29 phosphorylation sites.The secondary structure of SHR was mainly composed of alpha helix and the proportion of hydrophilic amino was over 60%.The antigenic epitopes prediction results indicated that SHR protein was a hydrophilic protein with high antigenicity.These results suggested that the SHR antigen has strong potential to be a candidate for immune diagnostics and used as a vaccine against P.skrjabini.
引文
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[17] 孙雪婧,杜晓华,杨孝朴,罗玉柱,刘霞.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698Sun X J,Du X H,Yang X P,Luo Y Z,Liu X.Cloning and bioinformatics analysis on CDS of CYGB gene in yak[J].Scientia Agricultura Sinica,2014,47(13):2690-2698 (in Chinese)
[18] 孙伟,李达,苏锐,马月辉,关伟军,张有法,陈玲,吴文忠,周洪.绵羊YAP1基因全长cDNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735Sun W,Li D,Su R,Ma Y H,Guan W J,Zhang Y F,Chen L,Wu W Z,Zhou H.Cloning and bioinformatics analysis of full-Length cDNA sequence of YAP1 gene in sheep[J].Scientia Agricultura Sinica,2013,46(8):1725-1735 (in Chinese)
[19] Li Y H,Liu X F,Zhu Y J,Zhou X T,Cao C B,Hu X A,Ma H M,Wen H,Ma X M,Ding J B.Bioinformatic prediction of epitopes in the Emy162 antigen of Echinococcus multilocularis[J].Experimental &Therapeutic Medicine,2013,6(2):335-340
[20] Zhang T,Li H,Lan X,Zhang C,Yang Z,Cao W,Fen N,Liu Y,Yan Y,Ya S A,Ma X.The bioinformatics analyses reveal novel antigen epitopes in major outer membrane protein of Chlamydia trachomatis[J].Indian Journal of Medical Microbiology,2017,35(4):522-528
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[22] Zhou J,Wang L,Zhou A,Lu G,Li Q H,Wang Z L,Zhu M Y,Zhou H Y,Cong H,He S Y.Bioinformatics analysis and expression of a novel protein ROP48 in Toxoplasma gondii[J].Acta Parasitologica,2016,61(2):319-328
[23] Cui J M,Zhang N Z,Li W H,Yan H B,Fu B Q.Cloning,identification,and bioinformatics analysis of a putative aquaporin TsAQP from Trichinella spiralis[J].Genetics & Molecular Research:GMR,2015,14(4):12699-12709
[24] 王文龙,赵学亮,孙柯,冯陈晨,王姝懿,王梦雅,刘春霞.斯氏副柔线虫CAMK/TSSK蛋白激酶基因的克隆及其蛋白质结构与抗原表位预测分析[J].畜牧兽医学报,2018,49(3):644-651Wang W L,Zhao X L,Sun K,Feng C C,Wang S Y,Wang M Y,Liu C X.Cloning,Protein structure analysis and antigenic epitopes prediction of immune-related gene,CAMK/TSSK protein kinase gene of Parabronema skrjabini[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(3):644-651 (in Chinese)
[2] 赵治国,杨晓野,杨莲茹,王瑞,王海艳,赵林立,敖威华,郭铁筝.内蒙古双峰驼斯氏副柔线虫病的流行病学调查[J].中国兽医科学,2012,42(12):1309-1314Zhao Z G,Yang X Y,Yang L R,Wang R,Wang H Y,Zhao L L,Ao W H,Guo T Z.Epidemiological investigation of Camelus bactrianus parabronemosis in Inner Mongolia[J].Chinese Veterinary Science,2012,42(12):1309-1314 (in Chinese)
[3] Mehlhor H.Encylopedic Reference of Parasitology[M].2nd ed.Berlin:Springer,2001:637-678
[4] Seyed S H.Molecular characterization of the first internal transcribed spacer of rDNA of Parabronema skrjabini for the first time in sheep[J].Annals of Parasitology,2015,61(4):241-246
[5] Hasheminasab S S,Jalousian F,Meshgi B.Molecular and morphological characterization of Parabronema skrjabini of sheep and goats at three different geographical zones in Iran[J].Annals of Parasitology,2016,62(1):55-61
[6] 杨莲茹,杨晓野,刘珍莲,张连生,张文彬.内蒙古地区骆驼斯氏副柔线虫病调查[J].内蒙古农业大学学报(自然科学版),2004,25(1):43-45Yang L R,Yang X Y,Liu Z L,Zhang L S,Zhang W B.Investigation of Parabronema skrjabini of camels in Inner Mongolia Region[J].Journal of Inner Mongolia Agricultural University,2004,25(1):43-45 (in Chinese)
[7] Alolea P J,Alonso A,Baugh L,Paisie C,Ramasamy G,Sekar A,Sur A,Jiménez M,Molina R,Larraga V,Myler P J.RNA-seq analysis reveals differences in transcript abundance between cultured and sand fly-derived Leishmania infantum promastigotes[J].Parasitology International,2018,67(4):476-480
[8] Zhu C H,Li X F,Zheng J Y.Transcriptome profiling using Illumina- and SMRT-based RNA-seq of hot pepper for in-depth understanding of genes involved in CMV infection[J].Gene,2018,666:123-133.
[9] 冯陈晨.斯氏副柔线虫转录组学和蛋白质组学研究及免疫相关基因鉴定[D].呼和浩特:内蒙古农业大学,2017Feng C C.Transcriptomic and proteomic analysis of Parabronema skrjabini and identification of immune-related genes[D].Hohhot:Inner Mongolia Agriculture University,2017 (in Chinese)
[10] Wilkins M R,Gasteiger E,Bairoch A,Sanchez J C,Willianms K L,Appel R D.Protein identification and analysis tools in the ExPASy server[J].Methods in Molecular Biology,1999,112:531-552
[11] Petersen T N,Brunak S,Heijne G V,Nielsen H.SignalP 4.1:discriminating signal peptides from transmembrane regions[J].Nature Methods,2011,8(10):785-786
[12] Blom N,Gammeltoft S,Brunak S.Sequence and structure-based prediction of eukaryotic protein phosphorylation sites[J].Journal of Molecular Biology,1999,294(5):1351-1362
[13] Moller S,Croning M D,Apweiler R.Evaluation of methods for the prediction of membrane spanning regions[J].Bioinformatics,2001,17(7):646-653
[14] Geourjon C,Deleage G.SOPMA:Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Computer Applications in the Biosciences,1995,11(6):681-684
[15] 杨波.角蝇生活习性及其斯氏副柔线虫感染情况的调查研究[D].呼和浩特:内蒙古农业大学,2014Yang B.Investigation of horn flies habits and their infection of Parabronema skrjabini[D].Huhhot:Inner Mongolia Agriculture University,2014 (in Chinese)
[16] Blazie S M,Babb C,Wilky H,Rawls A,Jin G P,Mangone M.Comparative RNA-Seq analysis reveals pervasive tissue-specific alternative polyadenylation in Caenorhabditis elegans intestine and muscles[J].BMC Biology,2015,13:4
[17] 孙雪婧,杜晓华,杨孝朴,罗玉柱,刘霞.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698Sun X J,Du X H,Yang X P,Luo Y Z,Liu X.Cloning and bioinformatics analysis on CDS of CYGB gene in yak[J].Scientia Agricultura Sinica,2014,47(13):2690-2698 (in Chinese)
[18] 孙伟,李达,苏锐,马月辉,关伟军,张有法,陈玲,吴文忠,周洪.绵羊YAP1基因全长cDNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735Sun W,Li D,Su R,Ma Y H,Guan W J,Zhang Y F,Chen L,Wu W Z,Zhou H.Cloning and bioinformatics analysis of full-Length cDNA sequence of YAP1 gene in sheep[J].Scientia Agricultura Sinica,2013,46(8):1725-1735 (in Chinese)
[19] Li Y H,Liu X F,Zhu Y J,Zhou X T,Cao C B,Hu X A,Ma H M,Wen H,Ma X M,Ding J B.Bioinformatic prediction of epitopes in the Emy162 antigen of Echinococcus multilocularis[J].Experimental &Therapeutic Medicine,2013,6(2):335-340
[20] Zhang T,Li H,Lan X,Zhang C,Yang Z,Cao W,Fen N,Liu Y,Yan Y,Ya S A,Ma X.The bioinformatics analyses reveal novel antigen epitopes in major outer membrane protein of Chlamydia trachomatis[J].Indian Journal of Medical Microbiology,2017,35(4):522-528
[21] Song J,He Q.Bioinformatics analysis of the structure and linear B-cell epitopes of aquaporin-3 from Schistosoma japonicum[J].Asian Pacific Journal of Tropical Medicine,2012,5(2):107-109
[22] Zhou J,Wang L,Zhou A,Lu G,Li Q H,Wang Z L,Zhu M Y,Zhou H Y,Cong H,He S Y.Bioinformatics analysis and expression of a novel protein ROP48 in Toxoplasma gondii[J].Acta Parasitologica,2016,61(2):319-328
[23] Cui J M,Zhang N Z,Li W H,Yan H B,Fu B Q.Cloning,identification,and bioinformatics analysis of a putative aquaporin TsAQP from Trichinella spiralis[J].Genetics & Molecular Research:GMR,2015,14(4):12699-12709
[24] 王文龙,赵学亮,孙柯,冯陈晨,王姝懿,王梦雅,刘春霞.斯氏副柔线虫CAMK/TSSK蛋白激酶基因的克隆及其蛋白质结构与抗原表位预测分析[J].畜牧兽医学报,2018,49(3):644-651Wang W L,Zhao X L,Sun K,Feng C C,Wang S Y,Wang M Y,Liu C X.Cloning,Protein structure analysis and antigenic epitopes prediction of immune-related gene,CAMK/TSSK protein kinase gene of Parabronema skrjabini[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(3):644-651 (in Chinese)
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