犬细小病毒VHH抗体的文库构建、表达及其鉴定

Library Construction,Expression and Identification of Canine Parvovirus VHH Antibody

关键词:  犬细小病毒  VHH抗体  噬菌体文库  真核表达

Key words:  canine parvovirus  VHH antibody  phage library  expression  identification

摘要:

为构建特异性犬细小病毒（Canine parvovirus，CPV）的纳米抗体库，并获得特异性抗CPV的VHH抗体。本试验以分离鉴定的一株CPV‐2c毒株（TS02株）作为免疫原免疫羊驼，四免后采集外周血，分离淋巴细胞，利用巢式PCR扩增VHH序列，通过噬菌体展示技术成功构建了纳米抗体的CPV免疫文库，进一步通过ELISA特异性筛选具有高亲和力的抗CPV纳米抗体序列。将筛选出的抗体序列插入pcDNA3.1真核表达载体构建pcDNA3.1‐VHH重组质粒，采用PEI转染高密度的HEK293F悬浮细胞，瞬时表达、纯化VHH，进行鉴定。结果表明，四次免疫后羊驼血清效价高达1:25000，达到了建库要求，构建的纳米抗体的CPV免疫文库，库容达2×106 CFU/mL，文库多样性良好。通过特异性筛选获得了4株具有高亲和力和特异性的抗CPV纳米抗体序列, 采用HEK293F细胞瞬时表达并纯化获得4个重组VHH，分别是CPV‐VHH‐H1、CPV‐VHH‐D4、CPV‐VHH‐F5和CPV‐VHH‐E3。ELISA和IFA试验结果表明表达出的4株重组纳米抗体均能够特异性结合CPV。本研究成功构建了CPV特异性纳米抗体文库，并筛选出4株特异性结合CPV的VHH抗体，为VHH抗体在犬细小的诊断和治疗方面的应用进行了初步的探索。

Abstract:

In order to construct a specific nanoantibody library of Canine parvovirus (CPV) and obtain specific VHH antibodies against CPV. A CPV‐2c strain (TS02 strain) isolated and identified was used as the immunogen for immunizing alpacas. After the fourth immunization, the peripheral blood was collected, then the lymphocytes were separated, and the VHH sequences were amplified by nested PCR. The CPV immune library of nanobodies was successfully constructed by phage display technology. The specific sequences of anti‐CPV nanobody with high affinity were further screened by ELISA. pcDNA3.1‐VHH recombinant plasmids were constructed by inserting the specific nanobody sequences into the pcDNA3.1 eukaryotic expression vector, then were transiently transfected into high-density HEK293F suspension cells with PEI. The recombinant VHHs were purified from the cell supernatant, then identified by ELISA and IFA. The results showed that the titer of alpaca serum was 1:25000 after four immunizations, which was qualified for library construction. The library capacity was 2×106 CFU/mL, indicated the library with good diversity. Four anti‐CPV nanobody sequences with high affinity and specificity were obtained by by transient expression and purification from HEK293F cells, namely CPV‐VHH‐H1, CPV‐VHH‐D4, CPV‐VHH‐F5 and CPV‐VHH‐E3. The results of ELISA and IFA test showed that all four recombinant nanobodies expressed could specifically bind CPV. The above results suggested that CPV‐specific nanolibraries were successfully constructed and four CPV‐specific VHH antibodies were screened out, which made a preliminary exploration for the application of VHH antibodies in the diagnosis and treatment of canine parvovirus infection.