狂犬病人源二硫键稳定单链抗体融合蛋白的构建及活性检测

摘要

[目的]构建、表达人源二硫键稳定单链抗体(scdsFv),检测其生物活性,以获得对狂犬病病毒有特异结合能力及中和活性的scdsFv蛋白.[方法]从GenBank上获得RV单抗SO57重链可变区VH和轻链可变区VL序列,在VH44和VL100位各突变一个氨基酸为半胱氨酸,用linker连接形成scdsFv,人工合成此序列,克隆人表达载体pET22b(+),在大肠杆菌中表达目的蛋白,镍柱亲和层析法纯化,并进行SDS-PAGE、Western blot鉴定.ELISA法和鼠脑组织抹片方法检测scdsFv对RV的特异结合活性;硫氰酸盐洗脱法测定scdsFv蛋白对RV的相对亲和力指数;分别用荧光抗体病毒中和试验(FAVN)和小鼠体内中和试验测定scdsFv的体外和体内中和活性.[结果]成功获得RV人源二硫键稳定单链抗体序列,大肠杆菌中表达得到scdsFv蛋白;分子量约为30.0kDa,Western blot表明此蛋白能与抗His单克隆抗体发生特异性反应.scdsFv能与RV Vero疫苗特异结合,且结合力随抗原浓度降低而降低;scdsFv能与鼠脑组织中的RV结合.FAVN法测得scdsFv的中和效价为41Iu/mL;小鼠体内中和试验表明scdsFv能保护55.6%鼠耐过强毒攻击.[结论]获得的scdsFv,具有良好的RV结合活性和体内外中和活性,有可能被用于暴露后狂犬病的预防.%[Objective]The purpose of this study is to produce new antibody molecules to neutralize the rabies virus specifically by the way of constructing and expressing the human anti-rabies virus scdsFv ( disulfide stabilized single chain antibody) gene,and characterizing its bioactivity.[Methods]We obtained the sequence of variable region of heavy chain (VH) and variable region of light chain ( VL) of RV monoclonal antibody SO57 from GenBank, and it respectively mutated into cysteine in the gene loci VH44 and VL100.The scdsFv gene was synthesized and inserted into a prokaryotic expression vector pET22b( + ).Purified inclusion body scdsFv proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay.The binding activity of scdsFv was identified by ELISA and mouse brain tissues infected with rabies virus CVS strain.The relative affinity of scdsFv was measured by ELISA using thiocyanate elution.The capacity of the scdsFv to neutralize the rabies virus CVS strains was determined by fluorescent antibody virus neutralisation test ( FAVN).The fusion proteins neutralized the CVS strain in a standard in vivo neutralized assay where the virus was incubated with the scdsFv molecules before intracranial inoculation in mice.[Results]The fragment genes of scdsFv to rabies virus were constructed successfully.ScdsFv protein was expressed in E.coli with approximate molecular weight of 30.0 kDa, which could be recognized by anti-His mAb.ELISA results demonstrated that scdsFv could bind antigen specificity.It was found that the strong reactivity of scdsFv to the smear of RV infected mouse-brain was demonstrated by IFA.As determined by FAVN with a reference serum,the titer of scdsFv was 41IU/mL.In addition, scdsFv could be 55.6％ protection of mice against lethal challenge with rabies virus CVS.[Conclusion]The scdsFv can bind antigen specificity and has neutralization capacity to the virus in vitro and in vivo.The anti-rabies scdsFv is potential for application in rabies post-exposure prophylaxis.