期刊文献+

人源抗狂犬病毒二硫键稳定性Fv抗体的生物学特性研究 被引量:7

Improved Biochemical Characteristics of Human Disulfide-stabilized Fv Fragment to Rabies Virus
下载PDF
导出
摘要 对抗狂犬病毒scFv进行稳定性改构 ,纯化制备有活性的人源抗狂犬病毒二硫键稳定性Fv抗体片段 (dsFv) ,然后对其生物学活性进行研究。将dsFvVH 、VL 基因在原核表达系统pET2 2b(+) BL2 1(DE3)中表达 ,将其包涵体分别在变性缓冲液中溶解 ,稀释后加入折叠缓冲液使其折叠形成有活性的dsFv抗体片段 ,上Ni NTA柱进行纯化。然后以其亲本scFv作为对照 ,对dsFv的亲和力、稳定性以及体外中和活性进行评价。结果显示 ,与其母本抗体scFv相比 ,改构后的抗狂犬病毒dsFv保持了对狂犬病毒Vero疫苗的特异性识别能力 ,而且其亲和力明显提高 ;抗狂犬病毒dsFv在血清和BSA中的稳定性有明显的改进 ,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进 ;蚀斑减少中和实验显示 ,抗狂犬病毒dsFv抗体片段能特异中和狂犬病毒 ,阻止狂犬病毒对Vero细胞的吸附 ,抑制狂犬病毒对靶细胞的感染 ,从而导致蚀斑减少甚至消失 ;scFv抗体片段仅可部分抑制蚀斑的形成 ,但不能全部抑制。这为进一步研究抗狂犬病毒dsFv基因工程抗体的免疫保护作用及其在临床的应用奠定了基础。 This study is (1) to improve the stabilization of human scFv to rabies virus; (2) to prepare active human dsFv fragment; and (3) to evaluate the biol ogical activities of dsFv. The dsFv V H and V L were separately expressed in P ET22 b(+)/BL21(DE3), solublized and combined in appropriate molar ratio in refolding solution. The resultant dsFv fragments were evaluated for its protection against rabies virus, its affinity and stability, in reference to the cognate scFv. Th e dsFv was found to bind specifically to Vero vaccine of rabies virus. Compared to the scFv, the dsFv was more stable, had higher affinity, and was able to inhi bit the infection of Rabies virus to Vero cell. This established a solid basis f or the clinical application of dsFv to rabies virus.
出处 《生物工程学报》 CAS CSCD 北大核心 2005年第2期265-269,共5页 Chinese Journal of Biotechnology
关键词 狂犬病毒 稳定性 二硫键稳定性Fv抗体 rabies virus, stability, disulfide-stabilized Fv
  • 相关文献

参考文献13

  • 1Huston JS, Levinson D, Mudgett HM et al. Protein engineering of antibody binding sites: recovery of specific activity in an antidoxigenin single-chain Fv analogue produced in Escherichia coli.Proc Natl Acad Sci USA, 1988, 85:5879
  • 2Brinkmann U, Reiter Y, Jung SH et al. A recombinant immunotoxin containing a disulfide-stabilized Fy fragment. Proc Natl Acad Sci USA, 1993, 90:7538 - 7542
  • 3Reiter Y, Brinkmann U, Webber K et al. Engineering interchain disulfide bonds into conserved framework regions of Fv fragments:improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv. Protein Eng, 1994, 7:697 - 704
  • 4Reiter Y, Brinkmann U, Lee B et al. Engineering antibody Fy fragments for cancer detection and therapy: disulfide-stabilized Fv fragments. Nature Biotech, 1996, 14:1239
  • 5赵小玲,荫俊,王慧,姜明,侯小军.人源抗狂犬病毒单链抗体库的构建及体外亲和筛选[J].细胞与分子免疫学杂志,2004,20(2):243-247. 被引量:11
  • 6赵小玲,荫俊.人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达[J].生物技术通讯,2004,15(2):115-117. 被引量:7
  • 7Webber KO, Reiter Y, Brinkmann U et al. Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog. Mol Immunol,1995, 32: 249
  • 8Colangeli R. Three-step purification of lipopolysaccaridefree polyhistidine-tagged recombinant antigens of Myobacterium tuberculosis. J Chromatography B, 1998, 714:223 - 235
  • 9Macdonald RA, Hosking CS, Jones CL. The measurement of relative affinity by ELISA using thiocyanate elution. J Immunol Methods, 1988, 106:191 - 194
  • 10Muller BH, Lafay F, Demangel C et al. Phage-displayed and soluble mouse scFv fragments neutralizes rabies virus. J Virol Methods, 1997, 67(2) :221 - 233

二级参考文献18

  • 1Champion JM,Kean RB,Rupprencht CE,et al.The development of monoclonal human rabies virus neurralizing antibodyies as a substitute for pooled human immune globulin in the prophylactic treatment of rabies virus esposure[J].J Immunol Methods,2000,235(1-2):81
  • 2Guo li,Yan Xiyun,Shijun,et al.Selecting and exspressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin[J].Biotechnol Appl Biochem,2000,31:21
  • 3Riechman L,Foote J,Winter G.Expression of an antibody Fv frahment in myeloma cells[J].J Mol Biol,1988,203(3):825
  • 4Kretschmar T,Aoustin L,Zingel O,et al.High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies[J] J Immunol Method,1996,195(1):93
  • 5Muller BH,Lafay F,Demangel C,et al.Phage-displayed and soluble mouse scFv fragments neutralizes rabies virus [J].J Virol Methods,1997,67(2):221
  • 6Dietzschold B.Antibody-mediated clearance of rabies virus from the mammalian central nervous system[J].Trends in Microbiol,1993,1:63
  • 7Wilkerson JA. Rabies update. Wilderness Environ [ J]. Medicine,2000, 11(1): 31 -39.
  • 8Cheung SC, Dietzschold B, Koprowski H, et al. A recombinant human Fab expressed in Escherichia colt neutralizes rabies virus [J]. JVirol, 1992, 66(11): 6714 -6720.
  • 9Muller B H, Lafay F, Demangel C, et al. Phage-displayed and soluble mouse scFv kagments neutralizes rabies virus [ J]. J Virol Methods, 1997, 67(2): 221 -233.
  • 10Nagesha HS, Wang LF, Shiell B, et al. A single chain Fy antibodydisplayed on phage surface recognises conformational group-specificepitope of bluetongue virus [ J ]. J Virol Methods, 2001 , 91 ( 2 ):203 - 207.

共引文献14

同被引文献68

引证文献7

二级引证文献11

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部