补体C9分子原核表达与多克隆抗体的制备
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摘要
通过大肠杆菌表达系统表达补体C9重组蛋白,并制备其多克隆抗体,用于检测沉积菌体表面的补体C9分子;方法:通过PCR以兔肝细胞cDNA文库为模板扩增获得补体C9部分基因,并将其克隆至原核表达载体pEASY~?-Blunt E1,经测序鉴定成功获得重组质粒pBlunt-C9。将其转化至大肠杆菌感受态细胞BL21(DE3)中,经IPTG诱导,Ni-NTA亲和层析柱纯化后,获得重组蛋白。将纯化的重组蛋白免疫BALB/c雌鼠,制备抗C9的鼠源多克隆抗体。结果显示,ELISA测得鼠抗C9多克隆抗体效价为1∶12 800,Western Blotting表明,制备的多克隆抗体可以特异性识别沉积在金黄色葡萄球菌和链球菌表面的C9分子,为进一步研究补体的生物学功能以及巩膜复合体(MAC)抗细菌感染机制奠定了基础。
In order to detect the complement C9 molecules on the surface of bacteria, the recombinant protein of complement C9 was expressed by E.coli expression system and polyclonal antibodies were prepared.The complement C9 gene was amplified from rabbit hepatocytes cDNA by PCR and cloned into the prokaryotic expression vector pEASY~?-Blunt E1. The recombinant plasmid pBlunt-C9 was confirmed to be successfully constructed by gene-sequencing and was transformed into the competent cell of E.coli BL21(DE3). Recombinant protein C9 was expressed by induced with IPTG, and purified by being Ni-NTA affinity chromatography column. BALB/c mice was immunized with the purified recombinant protein to prepare mouse polyclonal antibodies against C9.The polyclonal antibody showed a high titer of 1:12800 determined by ELISA and the C9 molecule precipitated on the surface of Staphyloccus aureus and Streptococcus bovis was detected by western blotting using the mouse anti-C9 polyclonal antibody, which laid a foundation for further study of the biological function of complement and MAC anti-bacterial infection mechanism.
In order to detect the complement C9 molecules on the surface of bacteria, the recombinant protein of complement C9 was expressed by E.coli expression system and polyclonal antibodies were prepared.The complement C9 gene was amplified from rabbit hepatocytes cDNA by PCR and cloned into the prokaryotic expression vector pEASY~?-Blunt E1. The recombinant plasmid pBlunt-C9 was confirmed to be successfully constructed by gene-sequencing and was transformed into the competent cell of E.coli BL21(DE3). Recombinant protein C9 was expressed by induced with IPTG, and purified by being Ni-NTA affinity chromatography column. BALB/c mice was immunized with the purified recombinant protein to prepare mouse polyclonal antibodies against C9.The polyclonal antibody showed a high titer of 1:12800 determined by ELISA and the C9 molecule precipitated on the surface of Staphyloccus aureus and Streptococcus bovis was detected by western blotting using the mouse anti-C9 polyclonal antibody, which laid a foundation for further study of the biological function of complement and MAC anti-bacterial infection mechanism.
引文
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[3] Schiela B,Bernklau S,Malekshahi Z,et al.Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex[J].Front Immunol,2018,9:2 177.
[4] 刘淑梅,官明德,李萍.血清补体C3、C4及高密度脂蛋白胆固醇与冠心病的关系研究[J].中国心血管病研究,2018,16(10):912- 915.
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[14] Hao Y,Zhao X,Yang J,et al.Monoclonal antibody targeting complement C9 binding domain of Trichinella spiralis paramyosin impairs the viability of Trichinella infective larvae in the presence of complement[J].Parasit Vectors,2014,7:313.
[15] Wang S,Wang R,Xu T.The evolutionary analysis on complement genes reveals that fishes C3 and C9 experience different evolutionary patterns[J].Fish Shellfish Immunol,2013,35(6):2 040- 2 045.
[16] Cantsilieris S,Nelson B J,Huddleston J,et al.Recurrent structural variation,clustered sites of selection,and disease risk for the complement factor H (CFH) gene family[J].Proc Natl Acad Sci U S A,2018,115(19):E4 433- E4 442.
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[19] Zhi-Min L.I.,张士璀.细菌逃避补体识别和攻击的机制[J].中国海洋大学学报(自然科学版),2008,38(4):595- 600,652.
[20] Wang Y,Bjes E S,Esser A F.Molecular aspects of complement-mediated bacterial killing.Periplasmic conversion of C9 from a protoxin to a toxin[J].J Biol Chem,2000,275(7):4 687- 4 692.
[21] Berends E T,Kuipers A,Ravesloot M M,et al.Bacteria under stress by complement and coagulation[J].FEMS Microbiol Rev,2014,38(6):1 146- 1 171.
[22] Tan L X,Toops K A,Lakkaraju A.Protective responses to sublytic complement in the retinal pigment epithelium[J].Proc Natl Acad Sci U S A,2016,113(31):8 789- 8 794.
[23] Napier B A,Brubaker S W,Sweeney T E,et al.Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity[J].J Exp Med,2016,213(11):2 365- 2 382.
[24] Stumpp N,Premnath P,Schmidt T,et al.Synthesis of new carolacton derivatives and their activity against biofilms of oral bacteria[J].Org Biomol Chem,2015,13(20):5 765- 5 774.
[25] Khanafer M,Al-Awadhi H,Radwan S.Coliform Bacteria for Bioremediation of Waste Hydrocarbons[J].Biomed Res Int,2017,2017:1 838 072.
[26] Alabresm A,Chen Y P,Decho A W,et al.A novel method for the synergistic remediation of oil-water mixtures using nanoparticles and oil-degrading bacteria[J].Sci Total Environ,2018,630:1 292- 1 297.
[2] Gorbushin A M.Immune repertoire in the transcriptome of Littorina littorea reveals new trends in lophotrochozoan proto-complement evolution[J].Dev Comp Immunol,2018,84:250- 263.
[3] Schiela B,Bernklau S,Malekshahi Z,et al.Active Human Complement Reduces the Zika Virus Load via Formation of the Membrane-Attack Complex[J].Front Immunol,2018,9:2 177.
[4] 刘淑梅,官明德,李萍.血清补体C3、C4及高密度脂蛋白胆固醇与冠心病的关系研究[J].中国心血管病研究,2018,16(10):912- 915.
[5] Bayly-Jones C,Bubeck D,Dunstone M A.The mystery behind membrane insertion:a review of the complement membrane attack complex[J].Philos Trans R Soc Lond B Biol Sci,2017,372(1726).
[6] Morgan B P,Boyd C,Bubeck D.Molecular cell biology of complement membrane attack[J].Semin Cell Dev Biol,2017,72:124- 132.
[7] Berends E T,Dekkers J F,Nijland R,et al.Distinct localization of the complement C5b- 9 complex on Gram- positive bacteria[J].Cell Microbiol,2013,15(12):1 955- 1 968.
[8] Witzel-Schlomp K,Rittner C,Schneider P M.The human complement C9 gene:structural analysis of the 5' gene region and genetic polymorphism studies[J].Eur J Immunogenet,2001,28(5):515- 522.
[9] Serna M,Giles J L,Morgan B P,et al.Structural basis of complement membrane attack complex formation[J].Nat Commun,2016,7:10 587.
[10] Zhao X,Hao Y,Yang J,et al.Mapping of the complement C9 binding domain on Trichinella spiralis paramyosin[J].Parasit Vectors,2014,7:80.
[11] Hepburn N J,Williams A S,Nunn M A,et al.In vivo characterization and therapeutic efficacy of a C5-specific inhibitor from the soft tick Ornithodoros moubata[J].J Biol Chem,2007,282(11):8 292- 8 299.
[12] Saar R M,Moskovich O,Iosefson O,et al.Mortalin/GRP75 binds to complement C9 and plays a role in resistance to complement-dependent cytotoxicity[J].J Biol Chem,2014,289(21):15 014- 15 022.
[13] Prasada R T,Lakshmi P T,Parvathy R,et al.Identification of second arginine-glycine-aspartic acid motif of ovine vitronectin as the complement C9 binding site and its implication in bacterial infection[J].Microbiol Immunol,2017,61(2):75- 84.
[14] Hao Y,Zhao X,Yang J,et al.Monoclonal antibody targeting complement C9 binding domain of Trichinella spiralis paramyosin impairs the viability of Trichinella infective larvae in the presence of complement[J].Parasit Vectors,2014,7:313.
[15] Wang S,Wang R,Xu T.The evolutionary analysis on complement genes reveals that fishes C3 and C9 experience different evolutionary patterns[J].Fish Shellfish Immunol,2013,35(6):2 040- 2 045.
[16] Cantsilieris S,Nelson B J,Huddleston J,et al.Recurrent structural variation,clustered sites of selection,and disease risk for the complement factor H (CFH) gene family[J].Proc Natl Acad Sci U S A,2018,115(19):E4 433- E4 442.
[17] Taylor R P,Lindorfer M A,Cook E M,et al.Hexamerization- enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9[J].Clin Immunol,2017,181:24- 28.
[18] Wright S D,Levine R P.How complement kills E.coli.I.Location of the lethal lesion[J].J Immunol,1981,127(3):1 146- 1 151.
[19] Zhi-Min L.I.,张士璀.细菌逃避补体识别和攻击的机制[J].中国海洋大学学报(自然科学版),2008,38(4):595- 600,652.
[20] Wang Y,Bjes E S,Esser A F.Molecular aspects of complement-mediated bacterial killing.Periplasmic conversion of C9 from a protoxin to a toxin[J].J Biol Chem,2000,275(7):4 687- 4 692.
[21] Berends E T,Kuipers A,Ravesloot M M,et al.Bacteria under stress by complement and coagulation[J].FEMS Microbiol Rev,2014,38(6):1 146- 1 171.
[22] Tan L X,Toops K A,Lakkaraju A.Protective responses to sublytic complement in the retinal pigment epithelium[J].Proc Natl Acad Sci U S A,2016,113(31):8 789- 8 794.
[23] Napier B A,Brubaker S W,Sweeney T E,et al.Complement pathway amplifies caspase-11-dependent cell death and endotoxin-induced sepsis severity[J].J Exp Med,2016,213(11):2 365- 2 382.
[24] Stumpp N,Premnath P,Schmidt T,et al.Synthesis of new carolacton derivatives and their activity against biofilms of oral bacteria[J].Org Biomol Chem,2015,13(20):5 765- 5 774.
[25] Khanafer M,Al-Awadhi H,Radwan S.Coliform Bacteria for Bioremediation of Waste Hydrocarbons[J].Biomed Res Int,2017,2017:1 838 072.
[26] Alabresm A,Chen Y P,Decho A W,et al.A novel method for the synergistic remediation of oil-water mixtures using nanoparticles and oil-degrading bacteria[J].Sci Total Environ,2018,630:1 292- 1 297.