中国明对虾ALFFc基因在毕赤酵母中的表达
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摘要
抗脂多糖因子(ALF)是一种抗菌活性小分子蛋白,在机体免疫系统中发挥重要作用,被认为具有替代抗生素药物的前景.该研究在已有中国明对虾ALFFc原核表达载体pET-DsbA-ALFFc的基础上,通过PCR在ALFFc的5’和3’端加上酶切位点,扩增到的片段与表达载体pPIC9K连接构建重组表达载体pPIC9K-ALFFc,转化到毕赤酵母GS115细胞中,通过筛选及鉴定获得甲醇利用型酵母转化子.通过甲醇诱导表达并镍柱纯化获得rALFFc,经SDS-PAGE和Western blot分析证明ALFFc在毕赤酵母中成功表达.
Anti-lipopolysaccharide factor(ALF)is a low molecular mass protein with antimicrobial activities and plays an important role in immune response against microbial invasion.Thus,they are considered to be good substitutes for traditional antibiotics.In the present study,the prokaryotic recombinant expression vector"pET-DsbA-ALFFc"was used as the template of PCR,which was constructed in previous study.EcoRI and NotI restriction sites were added to 5'and 3'ends of the ALFFc.The ALFFc fragment was ligated to pPIC9 K vector to construct a recombinant expression vector pPIC9 K-ALFFc,which then was transformed into competent Pichia pastoris GS115.Recombinant ALFFc(rALFFc)was induced with methanol and purified by Ni-chelating affinity chromatography.SDS-PAGE and Western blot analysis indicated that the rALFFc was expressed successfully in P.pastoris.
Anti-lipopolysaccharide factor(ALF)is a low molecular mass protein with antimicrobial activities and plays an important role in immune response against microbial invasion.Thus,they are considered to be good substitutes for traditional antibiotics.In the present study,the prokaryotic recombinant expression vector"pET-DsbA-ALFFc"was used as the template of PCR,which was constructed in previous study.EcoRI and NotI restriction sites were added to 5'and 3'ends of the ALFFc.The ALFFc fragment was ligated to pPIC9 K vector to construct a recombinant expression vector pPIC9 K-ALFFc,which then was transformed into competent Pichia pastoris GS115.Recombinant ALFFc(rALFFc)was induced with methanol and purified by Ni-chelating affinity chromatography.SDS-PAGE and Western blot analysis indicated that the rALFFc was expressed successfully in P.pastoris.
引文
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[5]TANAKA S,NAKAMURA T,MORITA T,et al.Limulus anti-LPS factor:an anticoagulant which inhibits the endotoxin mediated activation of Limulus coagulation system[J].Biochem Biophys Res Commun,1982,105(2):717-723.DOI:0006-291X(82)91493-0.
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[10]PONPRATEEP S,THARNTADA S,SOMBOONWIWAT K,et al.Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[J].Fish Shellfish Immunol,2012,32(1):26-34.DOI:10.1016/j.fsi.2011.10.010.
[11]JIANG H S,ZHANG Q,ZHAO Y R,et al.A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[J].Dev Comp Immunol,2015,48(1):33-42.DOI:10.1016/j.dci.2014.09.001.
[12]LIU J,YU Y,LI F,et al.A new anti-lipopolysaccharide factor(ALF)gene with its SNP polymorphisms related to WSSV-resistance of Litopenaeus vannamei[J].Fish Shellfish Immunol,2014,39(1):24-33.DOI:10.1016/j.fsi.2014.04.009.
[13]ROSA R D,STOCO P,HBARRACCO M A.Cloning and characterisation of cDNA sequences encoding for anti-lipopolysaccharide factors(ALFs)in Brazilian palaemonid and penaeid shrimps[J].Fish Shellfish Immunol,2008,25(5):693-696.DOI:10.1016/j.fsi.2008.08.001.
[14]LIU C C,CHUNG C P,LIN C Y,et al.Function of an anti-lipopolysaccharide factor(ALF)isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection[J].J Invertebr Pathol,2014,116:1-7.DOI:10.1016/j.jip.2013.12.001.
[15]REN Q,DU Z Q,LI M,et al.Cloning and expression analysis of an anti-lipopolysaccharide factor from giant freshwater prawn,Macrobrachium rosenbergii[J].Mol Biol Rep,2012,39(7):7673-7680.DOI:10.1007/s11033-012-1602-3.
[16]WANG Y,TANG T,GU J,et al.Identification of five anti-lipopolysaccharide factors in oriental river prawn,Macrobrachium nipponense[J].Fish Shellfish Immunol,2015,46(2):252-260.DOI:10.1016/j.fsi.2015.07.003.
[17]SUN C,XU W T,ZHANG H W,et al.An anti-lipopolysaccharide factor from red swamp crayfish,Procambarus clarkii,exhibited antimicrobial activities in vitro and in vivo[J].Fish Shellfish Immunol,2011,30(1):295-303.DOI:10.1016/j.fsi.2010.10.022.
[18]HOU Z G,WANG Y,HUI K,et al.A novel anti-lipopolysaccharide factor SpALF6in mud crab Scylla paramamosain exhibiting different antimicrobial activity from its single amino acid mutant[J].Dev Comp Immunol,2017,72:44-56.DOI:10.1016/j.dci.2017.02.009.
[19]YEDERY R,DREDDY K V.Identification,cloning,characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab,Scylla serrata[J].Fish Shellfish Immunol,2009,27(2):275-284.DOI:10.1016/j.fsi.2009.05.009.
[20]SHARMA S,YEDERY R D,PATGAONKAR M S,et al.Antibacterial activity of a synthetic peptide that mimics the LPS binding domain of Indian mud crab,Scylla serrata anti-lipopolysaccharide factor(SsALF)also involved in the modulation of vaginal immune functions through NF-kB signaling[J].Microb Pathog,2011,50(3-4):179-191.DOI:10.1016/j.micpath.2010.12.007.
[21]LIU H,JIRAVANICHPAISAL P,SODERHALL I,et al.Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus[J].J Virol,2006,80(21):10365-10371.DOI:10.1128/JVI.01101-06.
[22]LI C,ZHAO J,SONG L,et al.Molecular cloning,genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis[J].Dev Comp Immunol,2008,32(7):784-794.DOI:10.1016/j.dci.2007.11.008.
[23]ZHANG Y,WANG L,WANG L,et al.The second anti-lipopolysaccharide factor(EsALF-2)with antimicrobial activity fromEriocheir sinensis[J].Developmental&Comparative Immunology,2010,34(9):945-952.DOI:10.1016/j.dci.2010.04.002.
[24]LI X,CUI Z,LIU Y,et al.Polymorphisms of anti-lipopolysaccharide factors in the swimming crab Portunus trituberculatus and their association with resistance/susceptibility to Vibrio alginolyticus[J].Fish Shellfish Immunol,2013,34(6):1560-1568.DOI:10.1016/j.fsi.2013.03.373.
[25]LIU Y,CUI Z,LI X,et al.Molecular cloning,genomic structure and antimicrobial activity of PtALF7,a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus[J].Fish Shellfish Immunol,2013,34(2):652-659.DOI:10.1016/j.fsi.2012.12.002.
[26]柳峰松,李婷,王晓菲,等.中国明对虾ALFFc基因的原核表达与抗血清制备[J].河北大学学报(自然科学版),2010,30(1):88-92.DOI:10.3969/j.issn.1000-1565.2010.01.019.LIU F S,LI T,WANG X F,et al.Prokaryotic expression and antiserum preparation of ALFFc in Chinese shrimp(Fenneropenaeus chinensis)[J].Journal of Hebei University(Nature Science Edition),2010,30(1):88-92.DOI:10.3969/j.issn.1000-1565.2010.01.019.
[2]HANCOCK R E,BROWN K,LMOOKHERJEE N.Host defence peptides from invertebrates--emerging antimicrobial strategies[J].Immunobiology,2006,211(4):315-322.DOI:10.1016/j.imbio.2005.10.017.
[3]SILVA N C,SARMENTO BPINTADO M.The importance of antimicrobial peptides and their potential for therapeutic use in ophthalmology[J].Int J Antimicrob Agents,2013,41(1):5-10.DOI:10.1016/j.ijantimicag.2012.07.020.
[4]TASSANAKAJON A,SOMBOONWIWAT K,SUPUNGUL P,et al.Discovery of immune molecules and their crucial functions in shrimp immunity[J].Fish Shellfish Immunol,2013,34(4):954-967.DOI:10.1016/j.fsi.2012.09.021.
[5]TANAKA S,NAKAMURA T,MORITA T,et al.Limulus anti-LPS factor:an anticoagulant which inhibits the endotoxin mediated activation of Limulus coagulation system[J].Biochem Biophys Res Commun,1982,105(2):717-723.DOI:0006-291X(82)91493-0.
[6]YANG H,LI S,LI F,et al.Recombinant expression and functional analysis of an isoform of anti-lipopolysaccharide factors(FcALF5)from Chinese shrimp Fenneropenaeus chinensis[J].Dev Comp Immunol,2015,53(1):47-54.DOI:10.1016/j.dci.2015.06.015.
[7]YANG H,LI S,LI F,et al.Recombinant expression of a modified shrimp anti-lipopolysaccharide factor gene in Pichia pastoris GS115and its characteristic analysis[J].Mar Drugs,2016,14(8):152.DOI:10.3390/md14080152.
[8]TANG T,LI L,SUN L,et al.Functional analysis of Fenneropenaeus chinensis anti-lipopolysaccharide factor promoter regulated by lipopolysaccharide and(1,3)-beta-D-glucan[J].Fish Shellfish Immunol,2014,38(2):348-353.DOI:10.1016/j.fsi.2014.03.026.
[9]SOMBOONWIWAT K,MARCOS M,TASSANAKAJON A,et al.Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor(ALF)from the black tiger shrimp Penaeus monodon[J].Dev Comp Immunol,2005,29(10):841-851.DOI:10.1016/j.dci.2005.02.004.
[10]PONPRATEEP S,THARNTADA S,SOMBOONWIWAT K,et al.Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections[J].Fish Shellfish Immunol,2012,32(1):26-34.DOI:10.1016/j.fsi.2011.10.010.
[11]JIANG H S,ZHANG Q,ZHAO Y R,et al.A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response[J].Dev Comp Immunol,2015,48(1):33-42.DOI:10.1016/j.dci.2014.09.001.
[12]LIU J,YU Y,LI F,et al.A new anti-lipopolysaccharide factor(ALF)gene with its SNP polymorphisms related to WSSV-resistance of Litopenaeus vannamei[J].Fish Shellfish Immunol,2014,39(1):24-33.DOI:10.1016/j.fsi.2014.04.009.
[13]ROSA R D,STOCO P,HBARRACCO M A.Cloning and characterisation of cDNA sequences encoding for anti-lipopolysaccharide factors(ALFs)in Brazilian palaemonid and penaeid shrimps[J].Fish Shellfish Immunol,2008,25(5):693-696.DOI:10.1016/j.fsi.2008.08.001.
[14]LIU C C,CHUNG C P,LIN C Y,et al.Function of an anti-lipopolysaccharide factor(ALF)isoform isolated from the hemocytes of the giant freshwater prawn Macrobrachium rosenbergii in protecting against bacterial infection[J].J Invertebr Pathol,2014,116:1-7.DOI:10.1016/j.jip.2013.12.001.
[15]REN Q,DU Z Q,LI M,et al.Cloning and expression analysis of an anti-lipopolysaccharide factor from giant freshwater prawn,Macrobrachium rosenbergii[J].Mol Biol Rep,2012,39(7):7673-7680.DOI:10.1007/s11033-012-1602-3.
[16]WANG Y,TANG T,GU J,et al.Identification of five anti-lipopolysaccharide factors in oriental river prawn,Macrobrachium nipponense[J].Fish Shellfish Immunol,2015,46(2):252-260.DOI:10.1016/j.fsi.2015.07.003.
[17]SUN C,XU W T,ZHANG H W,et al.An anti-lipopolysaccharide factor from red swamp crayfish,Procambarus clarkii,exhibited antimicrobial activities in vitro and in vivo[J].Fish Shellfish Immunol,2011,30(1):295-303.DOI:10.1016/j.fsi.2010.10.022.
[18]HOU Z G,WANG Y,HUI K,et al.A novel anti-lipopolysaccharide factor SpALF6in mud crab Scylla paramamosain exhibiting different antimicrobial activity from its single amino acid mutant[J].Dev Comp Immunol,2017,72:44-56.DOI:10.1016/j.dci.2017.02.009.
[19]YEDERY R,DREDDY K V.Identification,cloning,characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab,Scylla serrata[J].Fish Shellfish Immunol,2009,27(2):275-284.DOI:10.1016/j.fsi.2009.05.009.
[20]SHARMA S,YEDERY R D,PATGAONKAR M S,et al.Antibacterial activity of a synthetic peptide that mimics the LPS binding domain of Indian mud crab,Scylla serrata anti-lipopolysaccharide factor(SsALF)also involved in the modulation of vaginal immune functions through NF-kB signaling[J].Microb Pathog,2011,50(3-4):179-191.DOI:10.1016/j.micpath.2010.12.007.
[21]LIU H,JIRAVANICHPAISAL P,SODERHALL I,et al.Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus[J].J Virol,2006,80(21):10365-10371.DOI:10.1128/JVI.01101-06.
[22]LI C,ZHAO J,SONG L,et al.Molecular cloning,genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis[J].Dev Comp Immunol,2008,32(7):784-794.DOI:10.1016/j.dci.2007.11.008.
[23]ZHANG Y,WANG L,WANG L,et al.The second anti-lipopolysaccharide factor(EsALF-2)with antimicrobial activity fromEriocheir sinensis[J].Developmental&Comparative Immunology,2010,34(9):945-952.DOI:10.1016/j.dci.2010.04.002.
[24]LI X,CUI Z,LIU Y,et al.Polymorphisms of anti-lipopolysaccharide factors in the swimming crab Portunus trituberculatus and their association with resistance/susceptibility to Vibrio alginolyticus[J].Fish Shellfish Immunol,2013,34(6):1560-1568.DOI:10.1016/j.fsi.2013.03.373.
[25]LIU Y,CUI Z,LI X,et al.Molecular cloning,genomic structure and antimicrobial activity of PtALF7,a unique isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus[J].Fish Shellfish Immunol,2013,34(2):652-659.DOI:10.1016/j.fsi.2012.12.002.
[26]柳峰松,李婷,王晓菲,等.中国明对虾ALFFc基因的原核表达与抗血清制备[J].河北大学学报(自然科学版),2010,30(1):88-92.DOI:10.3969/j.issn.1000-1565.2010.01.019.LIU F S,LI T,WANG X F,et al.Prokaryotic expression and antiserum preparation of ALFFc in Chinese shrimp(Fenneropenaeus chinensis)[J].Journal of Hebei University(Nature Science Edition),2010,30(1):88-92.DOI:10.3969/j.issn.1000-1565.2010.01.019.