摘要
为表达狂犬病病毒(Rabies Virus,RV)基质蛋白(M)蛋白,进而制备多克隆抗体。本研究以狂犬病病毒BD06株RNA为模版,通过RT-PCR扩增M蛋白基因全序列,将其插入质粒pFastbac I中,获取的重组质粒pFastbac I-M,经酶切鉴定后,转化到DH10Bac E.coli感受态中,以获取重组穿梭质粒Bacmid-M。用lipofectamine2000介导Bacmid-M转染Sf9细胞获取重组杆状病毒AcMNPV-M。用抗His标签的单体、犬抗RV阳性血清及兔抗RV阳性血清通过Western-Blot鉴定重组蛋白的表达及其反应原性。用镍离子亲和层析柱纯化重组蛋白,并免疫新西兰大耳白兔制备多克隆抗体,用Western-Blot和FAVN试验鉴定多克隆抗体的反应原性及其病毒中和效价。结果表明:(1)利用杆状病毒表达系统成功的表达了大小约为25kD的重组M蛋白;(2)重组M蛋白具有良好的免疫原性和反应原性;(3)制备的多克隆抗体与狂犬病病毒BD06、SRV 9、CVS-24、ERA、PV 2061及aG株的M蛋白均能发生特异性反应,但其无病毒中和能力。本研究完成了重组M蛋白的表达、纯化并制备了其多克隆抗体,为进一步研究M蛋白的生物学功能奠定了物质基础。
The purpose of this study was to express the matrix protein of rabies virus in baculovirus expression system and prepare its polyclonal antibody.Using the total RNA of RABV strain BD06 as a template,RT-PCR technique was utilized to amplify the sequence of M gene,which were then inserted into shuttle vector pFastbac I to construct the recombinant vector pFastbac I-M.After identification using the double restriction endonuclease cleavage method,the recombinant vector pFastbac I-M were transformed into the competent E.coli DH10 Bac to construct the recombinant expression vector Bacmid-M,which were transfected into Sf9 cells mediated by lipofectamine 2000 to obtain the recombinant baculovirus AcMNPV-M.The mice anti-His monoclonal antibody,rabbit anti-RV positive serum and canine anti-RV positive serum were used in Western Blot assays to identify the expression and reactogenicity of the recombinant.The recombinant M protein were purified under denaturing conditions using the nickel iron affinity chromatography column,then used to immunize the New Zealand White rabbit to prepare its polyclonal antibody.Western Blot assay and FAVN assay were used to validate the polyclonal antibody.Our results showed that the M protein of RABV were successfully expressed in baculovirus expression system,of which molecular weight was of about 25kD;the recombinant M protein has a good reactogenicity and immunogenicity;the rabbit polyclonal antibody prepared by purification of M protein could react with the M protein of RABV strain BD06,SRV 9,CVS-24,ERA,PV2061 and aG.Undoubtedly,the successfully preparation of both recombinant M protein and its polyclonal antibody support a material foundation for further study on the properties of M protein of RABV.
出处
《病毒学报》
CAS
CSCD
北大核心
2016年第4期472-477,共6页
Chinese Journal of Virology
基金
国家自然科学基金(31472176)
关键词
狂犬病病毒
基质蛋白
杆状病毒
表达
纯化
多克隆抗体
rabies virus
matrix protein
baculovirus
expression
purification
polyclonal antibody
