Preparation and identification of porcine XCR1/FMDV bispecific nanobody fusion protein
In order to enhance the immune efficacy of FMDV vaccine by targeting FMDV to porcine dendritic cells,the target protein XCR1 was expressed using prokaryotic expression technology.Subsequently,alpacas were immunized with XCR1 as an immunogen.The total mRNA of peripheral blood lymphocytes of alpacas was extracted and reverse-transcribed into cDNA.A phage display nanobody library was constructed and XCR1-specific nanobodies were obtained after three rounds of affinity screening.Following this,a bispecific nanobody Nb75-205 targeting both XCR1 and FMDV was constructed by connecting Nb75 with Nb205 anti-FMDV nanobody using SOE-PCR technique.The results showed that:1)The target protein XCR1 was successfully obtained,SDS-PAGE and Western blot analysis showed that the molecular weight of this protein was about 35 ku,as expected.2)Antibody titers of alpacas were 1:64 000,meeting the requirement for database construction.The phage display library size was approximately 1.91x108 CFU/mL,with an insertion rate of 91.3％and good diversity.After three rounds of affinity screening,the nanobody Nb75 was successfully obtained which could specifically bind to XCR1 as detected by Indirect ELISA.3)The XCR1/FMDV bispecific nanobody Nb75-205 was constructed through SOE-PCR technique.SDS-PAGE and Western blot results demonstrated that this fusion protein could be expressed in a soluble form in Escherichia coli,with a dimeric structure.Indirect ELISA results indicated that Nb75-205 was capable of binding to both XCR1 target protein and FMDV antigen.In summary,anti-porcine XCR1 nanobody was successfully obtained by phage screening technology,and XCR1/FMDV bispecific nanobody Nb75-205 was successfully constructed through SOE-PCR technology in this study.Nb75-205 was a dimer and could react with both XCR1 and FMDV,which laid a foundation for the further study on the presentation of FMDV targeted by porcine XCR1.
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