一株区分猪瘟病毒疫苗株和流行株的单克隆抗体的制备及其抗原表位鉴定

Preparation and antigenic epitope mapping of a monoclonal antibody capable of differentiating classical swine fever virus vaccine strain and field isolates

为制备可鉴别猪瘟病毒(CSFV)疫苗株(LPC)和流行株的单克隆抗体(MAb),并解析流行株逃逸宿主免疫血清中和抗体的机制,本研究利用猪瘟兔化弱毒疫苗株的E2蛋白免疫BALB/c小鼠,将免疫小鼠脾细胞和骨髓瘤细胞电融合,利用间接免疫荧光试验(IFA)筛选分泌抗CSFV E2蛋白MAb的杂交瘤细胞,利用IFA和western blot鉴定MAb与猪瘟兔化弱毒疫苗株及其不同基因型流行株或E2蛋白的反应原性以明确MAb的病毒反应谱。结果显示,本研究获得了一株仅与基因1.1和3.1亚型CSFV反应,但不与其他8个亚型流行株或E2蛋白反应的MAb TCH045,表明该MAb能够鉴别CSFV疫苗株和当前优势流行株。本实验进一步点突变E2蛋白后利用western blot对TCH045识别的抗原表位进行鉴定,结果显示,疫苗株E2蛋白的G^(19)、L^(21)、A^(23)、G^(24)和L^(26)突变后,TCH045与突变的E2蛋白不反应,表明这些氨基酸参与组成该MAb识别的抗原表位,其中G^(24)为关键氨基酸残基,而不与TCH045反应的流行株在该位点为E^(24)。此外,TCH045能够中和疫苗株(HCLV株和LPC株),但不能中和当前优势流行株,这可能是流行株逃逸免疫血清被中和的原因之一。综上所述,本研究获得了一株新的能区别CSFV疫苗株和流行株的中和MAb,可以用于疫苗免疫和流行株感染的血清学鉴别诊断技术的研发,初步揭示了CSFV流行株逃逸免疫血清中和的机制,为研发新型猪瘟疫苗提供了重要的科学数据。

To prepare monoclonal antibodies(MAbs)that can differentiate classical swine fever virus(CSFV)vaccine from field strains,the purified E2 protein derived from CSFV vaccine strain(LPC)was used to immunize BALB/c mice.Subsequently,mouse spleen cells were electronically fused with SP2/0 cells,and the hybridoma cells secreting CSFV specific MAbs were screened with PK-15 cells infected by vaccine strain using indirect immunofluorescence assay(IFA).The reaction spectrum of the obtained MAbs was further characterized through IFA and western blot with virus and/or E2 proteins belonging to different CSFV genotypes.As a result,MAb TCH045 was found to react with subgenotype 1.1 strains including vaccine strain and subgenotype 3.1 strain,but not with strains grouped into other 8 CSFV subgenotypes,indicating that TCH045 is capable of differentiating the CSFV vaccine strain from the currently dominant field strains.Furthermore,the antigenic epitope recognized by MAb TCH045 was identified through western blot.The epitope mapping results showed that LPC E2 protein with mutations at sitesG^(19)、L^(21)、A^(23)、G^(24) and L^(26) can not be recognized by MAb TCH045,indicating that these sites form the antigenic epitope of MAb TCH045 in E2 protein with G24 as the key amino acid residue,while the field strains containing E^(24) in E^(2 )proteins can not react with TCH045.In addition,MAb TCH045 is capable of neutralizing the vaccine strains HCLV and LPC,but not for dominant field strain,which partly explains why the field strains can escape the neutralization of the immunized sera induced by CSFV vaccine strains.Taken together,the obtained novel neutralizing MAb is capable of differentiating modified live vaccine from field isolates and can be used for the development of serological DIVA assay.Also,our study investigated the molecular mechanism of immune evasion of CSFV field isolates and the related results provide novel insights into the development of novel vaccine against CSF.