抗鹅星状病毒Cap蛋白单克隆抗体的制备及鉴定

自2016年以来,鹅星状病毒(GoAstV)已成为当前危害鹅养殖业生产的重要病原体之一,雏鹅均易感,主要引起9~19日龄雏鹅死亡,以雏鹅腿关节肿胀及肾脏尿酸盐沉积导致肾炎为主要病征。建立相应的病原学与血清学检测方法对于防控该病至关重要。为建立基于病毒Cap蛋白的诊断技术,本研究构建了鹅星状病毒去核定位信号肽的Cap基因原核表达质粒pET-30a-Cap,转化BL21(DE3)后用IPTG诱导表达,并通过SDS-PAGE和Western blot检测Cap蛋白的表达。大量表达并纯化重组蛋白后,将其免疫BALB/c小鼠,采用间接ELISA检测免疫小鼠抗体效价,制备单克隆抗体,并对其进行鉴定。结果显示:获得的重组蛋白分子量约为28 kDa;通过3次亚克隆筛选最终获得5株杂交瘤细胞株1A5G10、1C11B11、5B7G8、9A12B12、9C3E11,抗体效价均可达1:51~200以上;1A5G10、5B7G8、9C3E11重链为IgG1型,1C11B11、9A12B12重链为IgG2b型,轻链均为κ型;制备的单克隆抗体均可与重组蛋白反应或GoAstV发生反应;其中5B7G8株与GoAstV发生反应,1C11B11、9A12B12株与GoAstV发生反应。本研究结果为GoAstV诊断方法的建立以及GoAstV Cap蛋白功能的进一步研究奠定了坚实基础。

Preparation and Identification of Monoclonal Antibodies against Cap Protein for Goose Astrovirus

Goose astrovirus(GoAstV)has been one of the infectious agents for goslings since 2016.Goose astrovirus mainly infection causes illness,growth retardation and even deaths and characterized by uric acid salt deposits and gout in legs.The development of appropriate etiological and serological detection methods is essential to prevent and control the spread of the disease.In this study,the prokaryotic expression plasmid pET-30a-Cap containing Cap gene was constructed and transformed into BL21(DE3)for expression of the recombinant protein with IPTG induction.The expressed protein was examined in SDS-PAGE and Western blot.The results showed that the recombinant protein was mainly released into the culture supernatant and had a molecular mass of about 28 kDa as demonstrated in SDS-PAGE.The recombinant protein was then purified using His purification column.The purified protein was used to immunize BALB/cmice for preparation of monoclonal antibodies(mAbs).The immunized mice showed high ELISA titer(>51200)post-immunization and their spleen cells were fused with myeloma cells to get hybridomas.The hybridoma cells were cloned 3 times and 5 mAbs specific to Cap protein were obtained for further characterization.The isotyping of these mAbs revealed that 1A5G10,5B7G8 and 9C3E11 had the heavy chain type IgG1 and 1C11B11 and 9A12B12 had the heavy chain type IgG2b.All these MAbs had light chain typeκ.MAbs 5B7G8 reacted with GoAstV in Western blot and mAbs 1C11B11 and 9A12B12 reacted with GoAstV in both Western blot and immunofluorescence assay.The availability of these mAbs laid solid foundation for further development of diagnostic methods of GoAstV.