Construction Of A Phage Library Of Swine-Derived Single-Chain Antibody Against African Swine Fever Virus And Screening Of Single-Chain Antibodies

Single-chain antibody（ScFv）is a new type of genetically engineered antibody formed by linking the antibody heavy chain variable region（VH）and light chain variable region（VL）through a flexible polypeptide（Linker）.Compared with the monoclonal antibodies（MAb）,ScFv not only retains the specificity of the intact monoclonal antibody,but also has the characteristics of small molecular weight and low heterogeneity,making it a wide range of application prospects in targeted therapy and diagnostic testing.In this study,African swine fever virus（ASFV）was used as the research object to construct a pig-derived phage single-chain antibody library against ASFV,and then screening out a stable expression of pig-derived ScFv by testing with ASFV reactivity.ScFv can provide a new type of raw material for the diagnosis of ASFV.Firstly,PCR（SOE-PCR）primers was designed based on the porcine-derived antibody gene sequence published in the IMGT database,PBMCs from pig with ASFV antibody positive was isolated and the total RNA was extracted as a template for amplification.After two rounds of nested PCR with above primers,the pig-derived antibody VH and VL genes were obtained,respectively,and then through SOE-PCR,a linker sequence was used to connect VH and VL to form VH-Linker-VL,and the ScFv genes were obtained.Sequencing analysis revealed that the diversity of ScFv genes reaching more than 90%,which meets the requirements to build a database.Subsequently,the ScFv and the pHEN-2 phagemid vector were digested with restriction enzyme and ligated,the ligation product was then transformed into the TG1 host bacteria to obtain an original phage library with a capacity of 1.13×10⁸.The pig-derived ScFv phage library was obtained by means of the helper phage M13K07.The phage library was determined with a titer of 8.0×10¹³cfu/m L.Furthermore,the ASFV antigen was coated on detection plate,affinity enrichment of the phage library was performed,after 3 rounds of screening,a significantly enriched anti-ASFV ScFv phage library was obtained.Four specific ScFv strains against ASFV were screened out.Among them,VH-VLλ6 and VH-VLλ11 have good reactivity with ASFV.PCR amplification of VH-VLλ6 and VH-VLλ11 sequences,sequence comparison and analysis of NCBI Ig Blast sequence,the results confirmed that VH-VLλ6 and VH-VLλ11are from pig ScFv.Finally,VH-VLλ6 and VH-VLλ11 were constructed into p ET30a expression vector and transformed into BL21（DE3）competent cells for prokaryotic expression,respectively.Under the induction of IPTG,the expression product was analyzed by SDS-PAGE,and the size of target band was about 27KDa,and mainly existing in the form of inclusion bodies.The concentration of ScFv reaches more than 90%after purification by a nickel column.Furthermore,an enzyme-linked immunosorbent assay（ELISA）and an indirect immunofluorescence assay（IFA）were used to identify the reactivity between VH-VLλ6 and VH-VLλ11 with ASFV.The results demonstrated that the obtained two single-chain antibodies expressed by the prokaryotic expression system have good reactivity with ASFV.In summary,based on the SOE-PCR technology,the total RNA from the peripheral blood lymphocytes of the pigs naturally infected with ASFV was used as a template to successfully construct a porcine single-chain antibody phage library,and two ScFv were obtained from the library.Strain-specific ScFv against ASFV,further experiments proved that the two ScFv strains obtained by screening have ASFV reactivity.The successful preparation of this antibody fills the current gap in ASFV ScFv on the market and provides a new channel for the source of raw materials for ASFV diagnosis and prevention and control.