从江香猪IFN-β基因的序列分析及原核表达
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摘要
试验旨在探明从江香猪β-干扰素(interferon-beta,IFN-β)基因编码区分子序列及原核表达产物特征。以从江香猪为研究对象,提取肝脏总RNA并反转录为cDNA,设计特异性引物扩增IFN-β基因编码区,将目的基因片段克隆至原核表达质粒pET-28a上,获得重组质粒pET28a-CJpoIFN-β,并利用生物学软件对江香猪IFN-β基因编码区进行序列分析;将鉴定正确的重组质粒pET28a-CJpoIFN-β转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导表达、SDS-PAGE与Western blotting分析原核表达蛋白。结果表明,从江香猪IFN-β基因编码区长为561bp,编码186个氨基酸;该蛋白为分泌性蛋白,前21个氨基酸为信号肽序列;二级结构主要以α-螺旋(77.42%)和无规则卷曲(17.74%)为主。从江香猪与其他猪源IFN-β基因核苷酸序列同源性为99.5%～100.0%,与禽的同源性最低(35.2%);从江香猪与巴马猪、梅山猪IFN-β氨基酸同源性均为100.0%,但与贵州白香猪IFN-β同源性为99.5%,存在E43Q、K73R和C161R3处氨基酸的差异。Western blotting结果显示,带His标签的重组表达蛋白能被His单抗识别,条带大小约为24ku。本试验结果为进一步研究IFN-β基因生物学活性及加快从江香猪这一品种资源的有效利用提供参考依据。
This study was aimed to investigate the molecular sequences and prokaryotic expression product characteristics of interferon-beta(IFN-β)gene coding region in Congjiang Xiang pig.Congjiang Xiang pig was selected as the research object,the total RNA was extracted from liver tissue of Congjiang Xiang pig,and the mRNA was reversely transcribed into cDNA.The encoding region of IFN-βgene was amplified by specific primers,ligated into pET-28 avector,obtained the recombinant plasmid pET28a-CJpoIFN-β,and the CDS sequence of IFN-βgene was analyzed using bioinformatics softwares.The pET28a-CJpoIFN-β was transformed into Escherichia coli BL21(DE3)competent cells.After induction by IPTG,the product of prokaryotic expression was analyzed by SDS-PAGE and Western blotting.The results showed that the CDS sequence of IFN-βgene in Congjiang Xiang pig(CJpoIFN-β)was 561 bp,which encoded 186 amino acids;CJpoIFN-βprotein was secretory protein,and the first 21 amino acid residues was predicted to be the signal peptide.The secondary structure of CJpoIFN-βprotein was mainly alpha helix(77.42%)and random coli(17.74%).The nucleotide sequence alignment results showed that the IFN-βgene homology was 99.5%to 100.0%between Congjing Xiang pig and others porcine,the lowest homology was 35.2% between Congjing Xiang pig and poultry.Phylogenetic tree result showed that CJpoIFN-βwas closely related to that of Meishan pig and Bama pig,the amino acid homology was100.0%in Congjiang Xiang pig and Bama pig,Meishan pig;And the homology was 99.5% between Congjiang Xiang pig and Guizhou Baixiang pig,there were three amino acid differences in E43 Q,K73Rand C161 R.Western blotting results showed that His-tag recombinant protein could be recognized by His monoclonal antibody,the band was about 24 ku.The results of this experiment provided a reference for further study on the biological activity of IFN-βgene and speeding up the effective utilization of the resources of Congjiang Xiang pig.
This study was aimed to investigate the molecular sequences and prokaryotic expression product characteristics of interferon-beta(IFN-β)gene coding region in Congjiang Xiang pig.Congjiang Xiang pig was selected as the research object,the total RNA was extracted from liver tissue of Congjiang Xiang pig,and the mRNA was reversely transcribed into cDNA.The encoding region of IFN-βgene was amplified by specific primers,ligated into pET-28 avector,obtained the recombinant plasmid pET28a-CJpoIFN-β,and the CDS sequence of IFN-βgene was analyzed using bioinformatics softwares.The pET28a-CJpoIFN-β was transformed into Escherichia coli BL21(DE3)competent cells.After induction by IPTG,the product of prokaryotic expression was analyzed by SDS-PAGE and Western blotting.The results showed that the CDS sequence of IFN-βgene in Congjiang Xiang pig(CJpoIFN-β)was 561 bp,which encoded 186 amino acids;CJpoIFN-βprotein was secretory protein,and the first 21 amino acid residues was predicted to be the signal peptide.The secondary structure of CJpoIFN-βprotein was mainly alpha helix(77.42%)and random coli(17.74%).The nucleotide sequence alignment results showed that the IFN-βgene homology was 99.5%to 100.0%between Congjing Xiang pig and others porcine,the lowest homology was 35.2% between Congjing Xiang pig and poultry.Phylogenetic tree result showed that CJpoIFN-βwas closely related to that of Meishan pig and Bama pig,the amino acid homology was100.0%in Congjiang Xiang pig and Bama pig,Meishan pig;And the homology was 99.5% between Congjiang Xiang pig and Guizhou Baixiang pig,there were three amino acid differences in E43 Q,K73Rand C161 R.Western blotting results showed that His-tag recombinant protein could be recognized by His monoclonal antibody,the band was about 24 ku.The results of this experiment provided a reference for further study on the biological activity of IFN-βgene and speeding up the effective utilization of the resources of Congjiang Xiang pig.
引文
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[9]赵佳福,段志强,杨远青,等.从江香猪ApoA1基因的克隆及亚细胞定位研究[J].中国畜牧兽医,2017,44(7):1954-1960.ZHAO J F,DUAN Z Q,YANG Y Q,et al.Cloning and subcellular localization of ApoA1gene in Congjiang Xiang pig[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1954-1960.(in Chinese)
[10]曾芸,李军,赵武,等.广西巴马小型猪β-干扰素基因的克隆、序列分析和蛋白结构预测[J].河南农业科学,2007,36(5):105-108.ZENG Y,LI J,ZHAO W,et al.Cloning and sequence analysis of interferonβgene and its predicted protein structure in Bama Mini pig[J].Henan Agricultural Science,2007,36(5):105-108.(in Chinese)
[11]彭贵青,陈焕春,钱平.梅山猪β干扰素基因的克隆、原核表达及生物活性[J].中国兽医学报,2005,25(1):40-42.PENG G Q,CHEN H C,QIAN P.Cloning,expression and biological activity of Meishan porcine interferon beta gene[J].Chinese Journal of Veterinary Medicine,2005,25(1):40-42.(in Chinese)
[12]卜秀娟,胡仲明,柳巨雄,等.猪IFN-β的克隆与原核表达[J].中国兽医学报,2008,28(5):485-488.BU X J,HU Z M,LIU J X,et al.Molecular cloning of a novel porcine interferon-βand its prokaryotic expression[J].China Journal of Veterinary Medicine,2008,28(5):485-488.(in Chinese)
[13]温纳相,陈瑞爱,裴仉福,等.新兴猪干扰素-β基因的分子克隆与序列测定[J].动物医学进展,2005,26(5):77-79.WEN N X,CHEN R A,PEI Z F,et al.Cloning and sequencing of porcine IFN-β[J].Progress in Veterinary Medicine,2005,26(5):77-79.(in Chinese)
[14]HOLKO M,WILLIMS B R.Functional annotation of IFN-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines[J].Journal of Interferon and Cytokine Research,2006,26(8):534-547.
[15]KURATA K,IWATA A,MASUDA K,et al.Identification of CpG oligodeoxy nucleotide sequences that induce IFN-gamma production in canine peripheral blood mononu clear cells[J].Veterinary Immunology and Immunopathology,2004,102(4):441-450.
[16]刘华兰.牛β干扰素克隆、表达和鸡传染性支气管炎病毒受体鉴定[D].武汉:华中农业大学,2008.LIU H L.Cloning,prokaryotic expression of interferonβgene and construction of its eukaryotic expression vector in pichia pastoris and further identification of IBV receptoror[D].Wuhan:Huazhong Agricultural University,2008.(in Chinese)
[17]盖春云,张灿,单虎.水貂IFN-β基因的克隆与原核表达[J].动物医学进展,2009,46(6):39-43.GAI C Y,ZHANG C,SHAN H.Cloning and prokaryotic expression of IFN-βgene from mink[J].Progress in Veterinary Medicine,2009,46(6):39-43.(in Chinese)
[18]郑蓓.鸡和鹌鹑β-干扰素基因的克隆、表达及免疫调节作用的研究[D].泰安:山东农业大学,2014.ZHENG B.Cloning,prokaryotic expression of IFN-βof chicken andcoturnix and research of immune function[D].Tai’an:Shandong Agricultural University,2014.(in Chinese)
[19]苏凤艳,李哲,刘存发,等.梅花鹿IFN-β1的原核表达及活性鉴定[J].中国预防兽医学报,2015,37(2):136-139.SU F Y,LI Z,LIU C F,et al.Prokaryotic expression of interferon-bata 1of sika deer and the activity identification[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(2):136-139.(in Chinese)
[20]李林翔,程松,张晓明,等.华南虎和东北虎β-干扰素基因的克隆与遗传进化分析[J].畜牧与兽医,2015,47(12):80-83.LI L X,CHENG S,ZHANG X M,et al.Cloning and genetic evolution of interferonβgene from South China tiger and Amur tiger[J].Animal Husbandry&Veterinary Medicine,2015,47(12):80-83.(in Chinese)
[21]ARTURSSON K,GOBL A,LINDERSSON M,et al.Molecular cloning of a gene encoding porcine interferon-beta[J].Journal of Interferon Research,1992,12(3):153-160.
[22]孙成娟,许厚强,陈伟,等.贵州从江香猪FoxO4、PRKAA1基因在不同组织中的表达分析[J].基因组学与应用生物学,2017,3:841-846.SUN C J,XU H Q,CHEN W,et al.Analysis of the expression of FoxO4and PRKAA1gene in different tissues of Guizhou Congjiang Xiang pig[J].Genomics and Applied Biology,2017,3:841-846.(in Chinese)
[23]杨洋,许厚强,陈伟,等.贵州从江香猪PDK4、FGF10基因的克隆及组织表达分析[J].中国畜牧兽医,2017,44(12):3401-3409.YANG Y,XU H Q,CHEN W,et al.Cloning and tissue expression analysis of PDK4and FGF10gene from Congjiang Xiang pig in Guizhou province[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3401-3409.(in Chinese)
[24]陈雪梅,薛清华,祝荣格,等.猪Ⅰ型与Ⅱ型干扰素的克隆、表达及抗病毒活性比较[J].生物工程学报,2009,25(6):806-812.CHEN X M,XUE Q H,ZHU R G,et al.Cloning,expression and antiviral activity of porcine interferon typeⅠand typeⅡ[J].Chinese Journal of Biotechnology,2009,25(6):806-812.(in Chinese)
[25]白苏亚乐吐.小鼠IFN-β的原核表达及纯化[D].长春:吉林大学,2009.BAI S Y L T.Prokaryotic expression and purification of murine IFN-β[D].Changchun:Jilin University,2009.(in Chinese)
[2]陈诗军,陈余清.干扰素与肿瘤相关性研究进展[J].临床肺科杂志,2017,22(4):735-739.CHEN S J,CHEN Y Q.Advances in the relationship between interferon and tumor[J].Journal of Clinical Pulmonary Medicine,2017,22(4):735-739.(in Chinese)
[3]刘健,陈瑞爱,刘珊珊,等.猪α干扰素基因经密码子改造在毕赤酵母菌中的高效分泌表达[J].华南农业大学学报,2011,32(1):93-97.LIU J,CHEN R A,LIU S S,et al.High performance secreted expression of porcine interferonalhha gene in Pichia pastor is by codon change[J].Journal of South China Agricultural University,2011,32(1):93-97.(in Chinese)
[4]郑鸣,边传周,王老七,等.猪Ⅰ型干扰素融合表达及其抗病毒活性检测[J].中国兽医杂志,2012,48(6):43-46.ZHENG M,BIAN C Z,WANG L Q,et al.Study on the fusion expression and antiviral activity of porcine typeⅠinterferon[J].Chinese Journal of Veterinary Medicine,2012,48(6):43-46.(in Chinese)
[5]崔然.猪α、β、γ干扰素的表达及抗病毒活性的检测[D].泰安:山东农业大学,2012.CUI R.Expression and antiviral activity detection of porcine IFN-α,β,γ[D].Tai’an:Shandong Agricultural University,2012.(in Chinese)
[6]徐雷,冯若飞,李向茸,等.猪β干扰素真核表达载体pEGFP-C1-IFN-β的构建及在CHO细胞中的表达[J].中国兽医科学,2015,7:751-757.XU L,FENG R F,LI X R,et al.Construction of a eukaryotic expression vector pEGFP-C1-IFN-βfor porcine interferon beta and its expression in CHO cells[J].Chinese Veterinary Science,2015,7:751-757.(in Chinese)
[7]曲嘉琪,李洋,钟一鸣,等.β干扰素及其研究进展[J].安徽农业科学,2013,41(9):3792-3793.QU J Q,LI Y,ZHONG Y M,et al.Research progress of interferon beta[J].Journal of Anhui Agricultural Sciences,2013,41(9):3792-3793.(in Chinese)
[8]陈明飞,许厚强,陈伟,等.贵州从江香猪与中国农大小型猪Ⅰ系的杂交性能[J].贵州农业科学,2015,43(6):120-122.CHEN M F,XU H Q,CHEN W,et al.Crossing performance of Guizhou Congjiang Xiang pigⅠline of China Agricultural University Mini pig[J].Guizhou Agricultural Science,2015,43(6):120-122.(in Chinese)
[9]赵佳福,段志强,杨远青,等.从江香猪ApoA1基因的克隆及亚细胞定位研究[J].中国畜牧兽医,2017,44(7):1954-1960.ZHAO J F,DUAN Z Q,YANG Y Q,et al.Cloning and subcellular localization of ApoA1gene in Congjiang Xiang pig[J].China Animal Husbandry&Veterinary Medicine,2017,44(7):1954-1960.(in Chinese)
[10]曾芸,李军,赵武,等.广西巴马小型猪β-干扰素基因的克隆、序列分析和蛋白结构预测[J].河南农业科学,2007,36(5):105-108.ZENG Y,LI J,ZHAO W,et al.Cloning and sequence analysis of interferonβgene and its predicted protein structure in Bama Mini pig[J].Henan Agricultural Science,2007,36(5):105-108.(in Chinese)
[11]彭贵青,陈焕春,钱平.梅山猪β干扰素基因的克隆、原核表达及生物活性[J].中国兽医学报,2005,25(1):40-42.PENG G Q,CHEN H C,QIAN P.Cloning,expression and biological activity of Meishan porcine interferon beta gene[J].Chinese Journal of Veterinary Medicine,2005,25(1):40-42.(in Chinese)
[12]卜秀娟,胡仲明,柳巨雄,等.猪IFN-β的克隆与原核表达[J].中国兽医学报,2008,28(5):485-488.BU X J,HU Z M,LIU J X,et al.Molecular cloning of a novel porcine interferon-βand its prokaryotic expression[J].China Journal of Veterinary Medicine,2008,28(5):485-488.(in Chinese)
[13]温纳相,陈瑞爱,裴仉福,等.新兴猪干扰素-β基因的分子克隆与序列测定[J].动物医学进展,2005,26(5):77-79.WEN N X,CHEN R A,PEI Z F,et al.Cloning and sequencing of porcine IFN-β[J].Progress in Veterinary Medicine,2005,26(5):77-79.(in Chinese)
[14]HOLKO M,WILLIMS B R.Functional annotation of IFN-alpha-stimulated gene expression profiles from sensitive and resistant renal cell carcinoma cell lines[J].Journal of Interferon and Cytokine Research,2006,26(8):534-547.
[15]KURATA K,IWATA A,MASUDA K,et al.Identification of CpG oligodeoxy nucleotide sequences that induce IFN-gamma production in canine peripheral blood mononu clear cells[J].Veterinary Immunology and Immunopathology,2004,102(4):441-450.
[16]刘华兰.牛β干扰素克隆、表达和鸡传染性支气管炎病毒受体鉴定[D].武汉:华中农业大学,2008.LIU H L.Cloning,prokaryotic expression of interferonβgene and construction of its eukaryotic expression vector in pichia pastoris and further identification of IBV receptoror[D].Wuhan:Huazhong Agricultural University,2008.(in Chinese)
[17]盖春云,张灿,单虎.水貂IFN-β基因的克隆与原核表达[J].动物医学进展,2009,46(6):39-43.GAI C Y,ZHANG C,SHAN H.Cloning and prokaryotic expression of IFN-βgene from mink[J].Progress in Veterinary Medicine,2009,46(6):39-43.(in Chinese)
[18]郑蓓.鸡和鹌鹑β-干扰素基因的克隆、表达及免疫调节作用的研究[D].泰安:山东农业大学,2014.ZHENG B.Cloning,prokaryotic expression of IFN-βof chicken andcoturnix and research of immune function[D].Tai’an:Shandong Agricultural University,2014.(in Chinese)
[19]苏凤艳,李哲,刘存发,等.梅花鹿IFN-β1的原核表达及活性鉴定[J].中国预防兽医学报,2015,37(2):136-139.SU F Y,LI Z,LIU C F,et al.Prokaryotic expression of interferon-bata 1of sika deer and the activity identification[J].Chinese Journal of Preventive Veterinary Medicine,2015,37(2):136-139.(in Chinese)
[20]李林翔,程松,张晓明,等.华南虎和东北虎β-干扰素基因的克隆与遗传进化分析[J].畜牧与兽医,2015,47(12):80-83.LI L X,CHENG S,ZHANG X M,et al.Cloning and genetic evolution of interferonβgene from South China tiger and Amur tiger[J].Animal Husbandry&Veterinary Medicine,2015,47(12):80-83.(in Chinese)
[21]ARTURSSON K,GOBL A,LINDERSSON M,et al.Molecular cloning of a gene encoding porcine interferon-beta[J].Journal of Interferon Research,1992,12(3):153-160.
[22]孙成娟,许厚强,陈伟,等.贵州从江香猪FoxO4、PRKAA1基因在不同组织中的表达分析[J].基因组学与应用生物学,2017,3:841-846.SUN C J,XU H Q,CHEN W,et al.Analysis of the expression of FoxO4and PRKAA1gene in different tissues of Guizhou Congjiang Xiang pig[J].Genomics and Applied Biology,2017,3:841-846.(in Chinese)
[23]杨洋,许厚强,陈伟,等.贵州从江香猪PDK4、FGF10基因的克隆及组织表达分析[J].中国畜牧兽医,2017,44(12):3401-3409.YANG Y,XU H Q,CHEN W,et al.Cloning and tissue expression analysis of PDK4and FGF10gene from Congjiang Xiang pig in Guizhou province[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3401-3409.(in Chinese)
[24]陈雪梅,薛清华,祝荣格,等.猪Ⅰ型与Ⅱ型干扰素的克隆、表达及抗病毒活性比较[J].生物工程学报,2009,25(6):806-812.CHEN X M,XUE Q H,ZHU R G,et al.Cloning,expression and antiviral activity of porcine interferon typeⅠand typeⅡ[J].Chinese Journal of Biotechnology,2009,25(6):806-812.(in Chinese)
[25]白苏亚乐吐.小鼠IFN-β的原核表达及纯化[D].长春:吉林大学,2009.BAI S Y L T.Prokaryotic expression and purification of murine IFN-β[D].Changchun:Jilin University,2009.(in Chinese)
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