鸭补体调节因子H的原核表达及多克隆抗体制备
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摘要
为制备鸭补体调节因子H(CFH)的多克隆抗体,从健康雏鸭的肝脏组织中提取总RNA及反转录合成cDNA,PCR扩增补体调节因子H基因的2个片段H1和H2,插入到载体pMD19-T,测序正确后,将目的基因分别克隆至原核表达载体pCold-TF,转化至大肠埃希菌BL21(DE3),用IPTG诱导表达蛋白,重组蛋白经SDS-PAGE检测,镍柱亲和层析纯化的重组蛋白免疫新西兰大白兔,制备多克隆抗体。结果表明,成功构建重组表达质粒pCold-TF-H1和pCold-TF-H2,SDS-PAGE检测重组蛋白为可溶性表达,TF-H1和TF-H2多克隆抗体效价均超过1∶10 000。所制备的多克隆抗体可以为进一步研究鸭CFH的功能奠定基础。
In order to prepare polyclonal antibodies of duck complement regulatory factor H,total RNA was extracted from the liver of healthy ducklings.Two gene fragments of complement factor H(CFH),H1 and H2, were amplified by reverse transcription and PCR,and then were cloned into pMD19-T.After sequencing, the target genes were cloned into the prokaryotic expression vector pCold-TF,pCold-TF-H1 and pCold-TF-H2 were transformed into E.coli BL21(DE3),the expression proteins were induced by IPTG,detected by SDS-PAGE and conducted protein purification by nickel column affinity chromatography.The New Zealand white rabbits were immunized by purified TF-H1 and TF-H2 subcutaneously to prepare polyclonal antibodies.The results showed that the recombinant expression plasmids pCold-TF-H1 and pCold-TF-H2 were successfully constructed;the recombinant proteins were detected to be soluble by SDS-PAGE;the titers of polyclonal antibodies of both TF-H1 and TF-H2 exceeded 1∶10 000.The results indicated that the polyclonal antibodies would lay the foundation for further study of function of duck CFH.
In order to prepare polyclonal antibodies of duck complement regulatory factor H,total RNA was extracted from the liver of healthy ducklings.Two gene fragments of complement factor H(CFH),H1 and H2, were amplified by reverse transcription and PCR,and then were cloned into pMD19-T.After sequencing, the target genes were cloned into the prokaryotic expression vector pCold-TF,pCold-TF-H1 and pCold-TF-H2 were transformed into E.coli BL21(DE3),the expression proteins were induced by IPTG,detected by SDS-PAGE and conducted protein purification by nickel column affinity chromatography.The New Zealand white rabbits were immunized by purified TF-H1 and TF-H2 subcutaneously to prepare polyclonal antibodies.The results showed that the recombinant expression plasmids pCold-TF-H1 and pCold-TF-H2 were successfully constructed;the recombinant proteins were detected to be soluble by SDS-PAGE;the titers of polyclonal antibodies of both TF-H1 and TF-H2 exceeded 1∶10 000.The results indicated that the polyclonal antibodies would lay the foundation for further study of function of duck CFH.
引文
[1] Walport M J.Complement.Second of two parts[J].N Engl J Med,2001,344(15):1140-1144.
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[3] Grumach A A,Kirschfink M.Are complement deficiencies really rare?Overview on prevalence,clinical importance and modern diagnostic approach[J].Mol Immunol,2014,61(2):110-117.
[4] Pangburn M K,Schreiber R D,Muller-Eberhard H J.Human complement C3b inactivator:isolation,characterization,and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution[J].J Exp Med,1977,146(1):257-270.
[5] Weiler J M,DahaM R,Austen K F,et al.Control of the amplification convertase of complement by the plasma protein beta1H[J].Proc Natl Acad Sci USA,1976,73(9):3268-3272.
[6] Whaley K,Ruddy S.Modulation of the alternative complement pathways by beta 1 H globulin[J].J Exp Med,1976,144(5):1147-1163.
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[8] 杨汉春,郑世军,刘金华,等.动物免疫学[M].2版.北京:中国农业出版社,2003.
[9] 郭宝英,张川,祁鹏志,等.大黄鱼(Larimichthys crocea)补体调节因子CFH和CFHR2基因的分子特征及表达分析补体调节因子CFH和CFHR2基因的分子特征及表达分析[J].海洋与湖沼,2015,46(6):1406-1407.
[10] Meri T,Murgia R,Stefanel P,et al.Regulation of complement activation at the C3-level by serum resistant leptospires[J].Microb Pathog,2005,39(4):139-147.
[11] Barbosa A S,Abreu P A,Vasconcellos S A,et al.Immune evasion of Lleptospira species by acquisition of human complement regulator C4BP[J].Infect Immun,2009,77(3):1137-1143.
[12] Amdahl,Jarva H,Haanpera M,et al.Interactions between Bordetella pertussis and the complement inhibitor factor H[J].Mol Immunol,2011,48(4):697-705.
[13] Biedzka-Sarek M,Jarva H,Hyytiāinen H,et al.Characterization of complement factor H binding to Yersinia enterocolitica serotype O:3[J].Infect Immun,2008,76(9):4100-4109.
[14] Kajander T,Lehtinen M J,Hyv?rinen S,et al.Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement[J].Proc Natl Acad Sci USA,2011,108(7):2897-2902.
[15] Morgan H P,Schmidt C Q,Guariento M,et al.Structural basis for engagement by complement factor H of C3b on a self surface[J].Nat Struct Mol Biol,2011,18(4):463-470.
[2] Walport M J.Complement.First of two parts[J].N Engl J Med,2001,344 (14):1058-1066.
[3] Grumach A A,Kirschfink M.Are complement deficiencies really rare?Overview on prevalence,clinical importance and modern diagnostic approach[J].Mol Immunol,2014,61(2):110-117.
[4] Pangburn M K,Schreiber R D,Muller-Eberhard H J.Human complement C3b inactivator:isolation,characterization,and demonstration of an absolute requirement for the serum protein beta1H for cleavage of C3b and C4b in solution[J].J Exp Med,1977,146(1):257-270.
[5] Weiler J M,DahaM R,Austen K F,et al.Control of the amplification convertase of complement by the plasma protein beta1H[J].Proc Natl Acad Sci USA,1976,73(9):3268-3272.
[6] Whaley K,Ruddy S.Modulation of the alternative complement pathways by beta 1 H globulin[J].J Exp Med,1976,144(5):1147-1163.
[7] Lambris J D,Ricklin D,Geisbrecht B V.Complement evasion by human pathogens[J].Nat Rev Microbiol,2008,6(2):132-142.
[8] 杨汉春,郑世军,刘金华,等.动物免疫学[M].2版.北京:中国农业出版社,2003.
[9] 郭宝英,张川,祁鹏志,等.大黄鱼(Larimichthys crocea)补体调节因子CFH和CFHR2基因的分子特征及表达分析补体调节因子CFH和CFHR2基因的分子特征及表达分析[J].海洋与湖沼,2015,46(6):1406-1407.
[10] Meri T,Murgia R,Stefanel P,et al.Regulation of complement activation at the C3-level by serum resistant leptospires[J].Microb Pathog,2005,39(4):139-147.
[11] Barbosa A S,Abreu P A,Vasconcellos S A,et al.Immune evasion of Lleptospira species by acquisition of human complement regulator C4BP[J].Infect Immun,2009,77(3):1137-1143.
[12] Amdahl,Jarva H,Haanpera M,et al.Interactions between Bordetella pertussis and the complement inhibitor factor H[J].Mol Immunol,2011,48(4):697-705.
[13] Biedzka-Sarek M,Jarva H,Hyytiāinen H,et al.Characterization of complement factor H binding to Yersinia enterocolitica serotype O:3[J].Infect Immun,2008,76(9):4100-4109.
[14] Kajander T,Lehtinen M J,Hyv?rinen S,et al.Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement[J].Proc Natl Acad Sci USA,2011,108(7):2897-2902.
[15] Morgan H P,Schmidt C Q,Guariento M,et al.Structural basis for engagement by complement factor H of C3b on a self surface[J].Nat Struct Mol Biol,2011,18(4):463-470.
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