新蛭素和人IgG-Fc融合蛋白的表达纯化和功能评价
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摘要
新蛭素(EH)具有很好的抗凝血效果,但由于半衰期短限制了其在临床上的推广和应用.为延长EH的半衰期,制备人Ig G-Fc和EH的融合蛋白,并对其活性和药代动力学进行初步研究.将合成的Fc-EH和Fc-L-EH融合基因克隆至表达载体pc DNAHC上,再将重组表达载体转染CHO细胞.蛋白A亲和柱纯化融合蛋白,用SDSPAGE和WesternBlot鉴定蛋白表达情况,质谱法检测蛋白的分子质量和C端序列,体外凝块法和大鼠颈静脉血栓模型分别验证融合蛋白的体外抗凝活性和体内抗栓效果,应用化学发光免疫法检测融合蛋白半衰期.结果表明,成功构建了pc DNAHC-Fc-EH和pcDNAHC-Fc-L-EH两个重组表达载体,融合蛋白Fc-EH和Fc-L-EH分子质量分别为68 476,u和70 542,u,蛋白的C端序列正确,Fc-EH和Fc-L-EH活性分别为256,ATU/mg和64,ATU/mg.大鼠颈静脉血栓实验表明,Fc-EH具有抗血栓作用,体内消除半衰期为39.4,h.融合蛋白Fc-EH能延长EH体内半衰期,为融合蛋白的进一步研究奠定了基础.
Neorudin(EH)has a good anticoagulant effect.However,the short half-life of EH limits its clinical popularization and application.To prolong the half-life of EH,fusion proteins of Fc segment of human IgG and EH were prepared and the anticoagulant activity and pharmacokinetics of the fusion proteins were studied.The fusion genes FcEH and Fc-L-EH were cloned into the expression vector pcDNAHC and expressed in CHO cells.The fusion proteins were purified by protein A affinity chromatography and identified by SDS-PAGE and Western Blot.The molecular mass and C-terminal amino acid sequences of the fusion proteins were detected using mass spectrometry.The anticoagulant activities of fusion proteins in vitro and in vivo were detected with clot methods and jugular vein thrombosis rat models,respectively.The half-life of the fusion protein was tested in rats by chemiluminescence immunoassay.The results of restriction enzyme reaction indicated that the two recombinant expression vectors pcDNAHC-FcEH and pcDNAHC-Fc-L-EH were constructed,and the molecular masses of two targeting proteins were measured68,476,u and 70,542,u,respectively.The correct C-terminal amino acid sequences of the fusion proteins were obtained.The anticoagulant activity of the fusion protein Fc-EH is 256 ATU/mg,whereas that of Fc-L-EH is 64 ATU/mg.The results of jugular vein thrombosis rat models indicated that the fusion proteins of Fc-EH dosedependently inhibited thrombosis.In vivo pharmacokinetic,studies showed that the elimination half-life of Fc-EH spanned 39.4,h.These results demonstrate that the Fc-EH fusion protein prolongs the half-life of EH,paving the way for the further studies of its anticoagulant effects.
Neorudin(EH)has a good anticoagulant effect.However,the short half-life of EH limits its clinical popularization and application.To prolong the half-life of EH,fusion proteins of Fc segment of human IgG and EH were prepared and the anticoagulant activity and pharmacokinetics of the fusion proteins were studied.The fusion genes FcEH and Fc-L-EH were cloned into the expression vector pcDNAHC and expressed in CHO cells.The fusion proteins were purified by protein A affinity chromatography and identified by SDS-PAGE and Western Blot.The molecular mass and C-terminal amino acid sequences of the fusion proteins were detected using mass spectrometry.The anticoagulant activities of fusion proteins in vitro and in vivo were detected with clot methods and jugular vein thrombosis rat models,respectively.The half-life of the fusion protein was tested in rats by chemiluminescence immunoassay.The results of restriction enzyme reaction indicated that the two recombinant expression vectors pcDNAHC-FcEH and pcDNAHC-Fc-L-EH were constructed,and the molecular masses of two targeting proteins were measured68,476,u and 70,542,u,respectively.The correct C-terminal amino acid sequences of the fusion proteins were obtained.The anticoagulant activity of the fusion protein Fc-EH is 256 ATU/mg,whereas that of Fc-L-EH is 64 ATU/mg.The results of jugular vein thrombosis rat models indicated that the fusion proteins of Fc-EH dosedependently inhibited thrombosis.In vivo pharmacokinetic,studies showed that the elimination half-life of Fc-EH spanned 39.4,h.These results demonstrate that the Fc-EH fusion protein prolongs the half-life of EH,paving the way for the further studies of its anticoagulant effects.
引文
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[2]Sohn J,Kang H,Rao K,et al.Current status of the anticoagulant hirudin:Its biotechnological production and clinical practice[J].Applied Microbiology and Biotechnology,2001,57(5/6):606-613.
[3]Lubenow N,Greinacher A.Hirudin in heparin-induced thrombocytopenia[J].Seminars in Thrombosis and Hemostasis,2002,28(5):431-438.
[4]Nisio M D,Middeldorp S,Büller H R.Direct thrombin inhibitors[J].The New England Journal of Medicine,2005,353(10):1028-1040.
[5]Zhang C,Yu A,Yuan B,et al.Construction and functional evaluation of hirudin derivatives with low bleeding risk[J].Thromb Haemost,2008,99(2):324-330.
[6]王文文,徐向伟,赵专友.谷氨酸-脯氨酸-精氨酸-水蛭素抑制血栓形成的实验研究[J].中国药学杂志,2013,48(2):111-115.Wang Wenwen,Xu Xiangwei,Zhao Zhuanyou,et al.Anti-thrombus activity of a novel anti-thrombus protein EPR-hirudin[J].Chinese Pharmaceutical Journal,2013,48(2):111-115(in Chinese).
[7]Dong Xiaona,Meng Zhiyun,Jin Jide.Development,validation,and clinical pharmacokinetic application of ultraperformance liquid chromatography/tandem mass spectrometry method for simultaneously determining a novel recombinant hirudin derivative(neorudin)and its active metabolite in human serum[J].Journal of Chromatography B,2017,1063:204-213.
[8]Jonathan T S,Szoka F C.The neonatal Fc receptor,FcRn,as a target for drug delivery and therapy[J].Adv Drug Deliv Rev,2015,91(30):109-124.
[9]Chen X Y,Zaro J L,Shen W C.Fusion protein linkers:Property,design and functionality[J].Adv Drug Deliv Rev,2013,65(10):1357-1369.
[10]Czajkowsky D M,Hu J,Shao Z,et al.Fc-fusion proteins:New developments and future perspectives[J].EMBO Mol Med,2012,4(10):1015-1028.
[11]秦晓永,于爱平,毕建进.抗凝蛋白EH体外活性检测条件的建立[J].中国生物工程杂志,2010,31(5):108-112.Qin Xiaoyong,Yu Aiping,Bi Jianjin.The anticoagulant activity detection of EH in vitro[J].China Biotechnology,2010,31(5):108-112(in Chinese).
[12]Van Witteloostuijn S B,Pedersen S L,Jensen K J.Half-life extension of biopharmaceuticals using chemical methods:Alternatives to PEGylation[J].ChemMedChem,2016,11(22):2474-2495.
[13]Jevsevar S,Kunstelj M,Porekar V G.PEGylation of therapeutic proteins[J].Biotechnol J,2010,5(1):113-128.
[14]Caliceti P,Veronese F M.Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates[J].Advanced Drug Delivery Reviews,2003,55(10):1261-1277.
[15]Strohl W R.Fusion proteins for half-life extension of biologics as a strategy to make biobetters[J].Biodrugs,2015,29(4):215-239.
[16]Rath T,Baker K,Dumont J A,et al.Fc-fusion proteins and FcRn:Structural insights for longer-lasting and more effective therapeutics[J].Crit Rev Biotechnol,2015,35(2):235-254.
[17]Roopenian D C,Akilesh S.FcRn:The neonatal Fc receptor comes of age[J].Nat Rev Immunol,2007,7(9):715-725.
[18]Kontermann R E.Strategies for extended serum half-life of protein therapeutics[J].Curr Opin Biotechnol,2011,22(6):868-876.
[19]Kontermann R E.Strategies to extend plasma half-lives of recombinant antibodies[J].Biodrugs,2009,23(2):93-109.
[20]Strohl W R.Current progress in innovative engineered antibodies[J].Protein Cell,2017,9(1):86-120.
[21]Zhao H L,Yao X Q,Xue C,et al.Increasing the homogeneity,stability and activity of human serum albumin and interferon-?2b fusion protein by linker engineering[J].Protein Expression and Purification,2008,61(1):73-77.