猪带绦虫丝氨酸蛋白酶抑制剂4848的鉴定及表达
|
摘要
为鉴定并表达猪带绦虫丝氨酸蛋白酶抑制剂(TsSerpin),提取猪囊尾蚴总RNA,设计引物并通过RT-PCR技术扩增TsSerpin4848基因,将纯化的PCR产物连接到p MD19-T载体后转化大肠杆菌DH5ɑ感受态细胞,筛选阳性克隆并测序,并用生物信息学方法分析其序列和结构特征。构建p ET-30a(+)-4848重组表达载体,经酶切鉴定及测序验证后转入大肠杆菌BL21(DE3)中进行诱导表达,目的蛋白纯化后进行SDSPAGE和Western-blot分析。结果表明,TsSerpin4848的开放阅读框由1 065个核苷酸组成,编码355个氨基酸残基,其理论分子质量约为39 ku。TsSerpin4848含有serpin家族保守序列DEEGAE和FIVDHPFLFFI,并含有serpin特有的反应中心环结构域,其三级结构中有8个ɑ螺旋和3个β折叠;重组TsSerpin4848蛋白在大肠杆菌中主要以包涵体形式存在,可被猪囊尾蚴阳性血清识别,表明TsSerpin4848蛋白具有良好的反应原性,有望作为猪囊虫病诊断的候选抗原。
To identify and express the serine protease inhibitor of Taenia solium(TsSerpin),the total RNA was extracted from T.solium metacestodes.Specific primers were designed and used to amplify TsSerpin4848 by RT-PCR.PCR products were ligated into the p MD19-T vector.After being transformed into Escherichia coli DH5ɑ,the positive clones were identified and confirmed by sequencing.The sequence of TsSerpin4848 was analyzed with the bioinformatics software.The recombinant p ET-30 a(+)-4848 plasmid was constructed and transformed into E.coli BL21(DE3).The target protein was expressed,purified and analyzed by SDS-PAGE and Western-blot.In result,TsSerpin4848 c DNA sequence contained an open reading frame of 1 065 nucleotides,and the deduced protein consisted of 355 amino acids with the theoretical protein molecular weight of 39 ku.TsSerpin4848 had a serpin conserved structure domain with the DEEGAE and FIVDHPFLFFI,and a specific reactive centre loop(RCL) motif.Analysis of tertiary structure of TsSerpin-4848 revealed that it had eight α-helices and three β-sheets.The expressed products of TsSerpin4848,existed in inclusion bodies,could be recognized by the positive serum from the pig infected with T.solium by immunoblotting,indicating that the protein TsSerpin4848 has a good immunogenicity and it is hopeful to be a diagnostic antigen for cysticercosis.
To identify and express the serine protease inhibitor of Taenia solium(TsSerpin),the total RNA was extracted from T.solium metacestodes.Specific primers were designed and used to amplify TsSerpin4848 by RT-PCR.PCR products were ligated into the p MD19-T vector.After being transformed into Escherichia coli DH5ɑ,the positive clones were identified and confirmed by sequencing.The sequence of TsSerpin4848 was analyzed with the bioinformatics software.The recombinant p ET-30 a(+)-4848 plasmid was constructed and transformed into E.coli BL21(DE3).The target protein was expressed,purified and analyzed by SDS-PAGE and Western-blot.In result,TsSerpin4848 c DNA sequence contained an open reading frame of 1 065 nucleotides,and the deduced protein consisted of 355 amino acids with the theoretical protein molecular weight of 39 ku.TsSerpin4848 had a serpin conserved structure domain with the DEEGAE and FIVDHPFLFFI,and a specific reactive centre loop(RCL) motif.Analysis of tertiary structure of TsSerpin-4848 revealed that it had eight α-helices and three β-sheets.The expressed products of TsSerpin4848,existed in inclusion bodies,could be recognized by the positive serum from the pig infected with T.solium by immunoblotting,indicating that the protein TsSerpin4848 has a good immunogenicity and it is hopeful to be a diagnostic antigen for cysticercosis.
引文
[1]AKIRA I,CARLO U,QIU J M,et al.Control of echinococcosis and cysticercosis:a public health challenge to international cooperation in China[J].Acta Trop,2003,86(1):3-17.
[2]CORAL-ALMEIDA M,GABRIEL S,ABATIH E N,et al.Taenia solium human cysticercosis:a systematic review of sero-epidemiological data from endemic zones around the world[J].PLo S Negl Trop Dis,2015,9(7):e0003919.
[3]SALZET M,VIEAU D,STEFANO G B.Serpins:an evolutionarily conserved survival strategy[J].Immunol Today,1999,20(12):541-544.
[4]GETTINS P G.The F-helix of serpins plays an essential,active role in the proteinase inhibition mechanism[J].FEBS Lett,2002,523(1-3):2-6.
[5]GETTINS P G W,OLSON S T.Exosite determinants of serpin specificity[J].J Biol Chem,2009,284(31):20441-20445.
[6]IRVING J A,PIKE R N,LESK A M,et al.Phylogeny of the serpin superfamily:implications of patterns of amino acid conservation for structure and function[J].Genome Res,2000,10(12):1845-1864.
[7]SUGINO M,IMAMURA S,MULENGA A,et al.A serine proteinase inhibitor(serpin)from ixodid tick Haemaphysalis longicornis;cloning and preliminary assessment of its suitability as a candidate for a tick vaccine[J].Vaccine,2003,21(21):2844-2851.
[8]CONGOTE L F.Serpin A1 and CD91 as host instruments against HIV-1 infection:Are extracellular antiviral peptides acting as intracellular messengers[J].Virus Res,2007,125(2):119-134.
[9]THORGERSEN E B,GHEBREMARIAM Y T,THURMAN J M,et al.Candidate inhibitors of porcine complement[J].Mol Immunol,2007,44(8):1827-1834.
[10]GHENDLER Y,ARNON R,FISHELSON Z.Schistosoma mansoni:isolation and characterization of Smpi56,a novel serine protease inhibitor[J].Exp Parasitol,1994,78(2):121-131.
[11]LIM K C,SUN E,BAHGAT M,et al.Blockage of skin invasion by schistosome cercariae by serine protease inhibitors[J].Am JTrop Med Hyg,1999,60(3):487-492.
[12]MERCKELBACH A,RUPPEL A.Biochemical properties of an intracellular serpin from Echinococcus multilocularis[J].Mol Biochem Parasitol,2007,156(1):84-88.
[13]WHISSTOCK J,SKINNER R,LESK A M.An atlas of serpin conformations[J].Trends Biochem Sci,1998,23(2):63-67.
[14]IRVING J A,ASKEW D J,WHISSTOCK J C.Computational analysis of evolution and conservation in a protein superfamily[J].Methods,2004,32(2):73-92.
[15]YAN Y T,LIU S X,SONG G C,et al.Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum(Sjserpin)[J].Vet Parasitol,2005,131(1-2):53-60.
[16]NAGANO I,WU Z,NAKADA T,et al.Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis[J].Parasitology,2001,123(1):77-83.
[17]吴秀萍,赫荣华,邹洪斌,等.旋毛虫肌幼虫强反应原性抗原基因的筛选及生物学特性分析[J].吉林农业大学学报,2008,30(2):213-218.WU Xiu-ping,HE Rong-hua,ZOU Hong-bin,et al.Immunoscreening and characterization analysis of strong reactive antigenicity genes of muscle larvae stage of Trichinella spiralis[J].Journal of Jilin Agricultural University,2008,30(2):213-218.(in Chinese)
[18]盖文燕,李文卉,王鸿盛,等.旋毛虫丝氨酸蛋白酶抑制因子基因的克隆与原核表达[J].中国兽医科学,2010,40(5):470-474.GE Wen-yan,LI Wen-hui,WANG Hong-sheng,et al.Cloning and prokaryotic expression of serine proteinase inhibitor gene of Trichinella spiralis[J].Chinese Veterinary Science,2010,40(5):470-474.(in Chinese)
[2]CORAL-ALMEIDA M,GABRIEL S,ABATIH E N,et al.Taenia solium human cysticercosis:a systematic review of sero-epidemiological data from endemic zones around the world[J].PLo S Negl Trop Dis,2015,9(7):e0003919.
[3]SALZET M,VIEAU D,STEFANO G B.Serpins:an evolutionarily conserved survival strategy[J].Immunol Today,1999,20(12):541-544.
[4]GETTINS P G.The F-helix of serpins plays an essential,active role in the proteinase inhibition mechanism[J].FEBS Lett,2002,523(1-3):2-6.
[5]GETTINS P G W,OLSON S T.Exosite determinants of serpin specificity[J].J Biol Chem,2009,284(31):20441-20445.
[6]IRVING J A,PIKE R N,LESK A M,et al.Phylogeny of the serpin superfamily:implications of patterns of amino acid conservation for structure and function[J].Genome Res,2000,10(12):1845-1864.
[7]SUGINO M,IMAMURA S,MULENGA A,et al.A serine proteinase inhibitor(serpin)from ixodid tick Haemaphysalis longicornis;cloning and preliminary assessment of its suitability as a candidate for a tick vaccine[J].Vaccine,2003,21(21):2844-2851.
[8]CONGOTE L F.Serpin A1 and CD91 as host instruments against HIV-1 infection:Are extracellular antiviral peptides acting as intracellular messengers[J].Virus Res,2007,125(2):119-134.
[9]THORGERSEN E B,GHEBREMARIAM Y T,THURMAN J M,et al.Candidate inhibitors of porcine complement[J].Mol Immunol,2007,44(8):1827-1834.
[10]GHENDLER Y,ARNON R,FISHELSON Z.Schistosoma mansoni:isolation and characterization of Smpi56,a novel serine protease inhibitor[J].Exp Parasitol,1994,78(2):121-131.
[11]LIM K C,SUN E,BAHGAT M,et al.Blockage of skin invasion by schistosome cercariae by serine protease inhibitors[J].Am JTrop Med Hyg,1999,60(3):487-492.
[12]MERCKELBACH A,RUPPEL A.Biochemical properties of an intracellular serpin from Echinococcus multilocularis[J].Mol Biochem Parasitol,2007,156(1):84-88.
[13]WHISSTOCK J,SKINNER R,LESK A M.An atlas of serpin conformations[J].Trends Biochem Sci,1998,23(2):63-67.
[14]IRVING J A,ASKEW D J,WHISSTOCK J C.Computational analysis of evolution and conservation in a protein superfamily[J].Methods,2004,32(2):73-92.
[15]YAN Y T,LIU S X,SONG G C,et al.Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum(Sjserpin)[J].Vet Parasitol,2005,131(1-2):53-60.
[16]NAGANO I,WU Z,NAKADA T,et al.Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis[J].Parasitology,2001,123(1):77-83.
[17]吴秀萍,赫荣华,邹洪斌,等.旋毛虫肌幼虫强反应原性抗原基因的筛选及生物学特性分析[J].吉林农业大学学报,2008,30(2):213-218.WU Xiu-ping,HE Rong-hua,ZOU Hong-bin,et al.Immunoscreening and characterization analysis of strong reactive antigenicity genes of muscle larvae stage of Trichinella spiralis[J].Journal of Jilin Agricultural University,2008,30(2):213-218.(in Chinese)
[18]盖文燕,李文卉,王鸿盛,等.旋毛虫丝氨酸蛋白酶抑制因子基因的克隆与原核表达[J].中国兽医科学,2010,40(5):470-474.GE Wen-yan,LI Wen-hui,WANG Hong-sheng,et al.Cloning and prokaryotic expression of serine proteinase inhibitor gene of Trichinella spiralis[J].Chinese Veterinary Science,2010,40(5):470-474.(in Chinese)