The Preparation Of A Single Chain Antibody Varible Fragment Against The Polar Flagellin B In Vibrio Parahaemolyticus

Vibrio parahaemolyticus,a Gram-negative , halophilic bacterium that inhabits in the marine and estuarine environments , is a major foodborne pathogen that causes diarrheain human through seafood consumption,and polluted waterfood. Its flagellar gene system is very complicated, involved in adhesion, including two forms such as the whole body flagella and the polar flagella. The flagella system have 57 genes and 3 ORFs, polar flagella system does not share the structure and assembly of components with the whole body flagella system.In this study , rapid and accurate methods for detection for Vibrio parahaemolyticus strains were established based on immunoflurescenced scFv of the polar flagellin FlaB. First, VP flaB gene was amplified by PCR from VP genome, and the fusion expression vector pET-32a(+)-flaB was constructed, which then expressed the fusion protein in E.coli BL21 by induced by IPTG. The protein was purified with Ni2+-NTA affinity chromatography.Immunizated mice BalB/C with the protein FlaB, tested titer of serum, Extracted the spleen RNA ,reverse transcription–PCR the heavy chain variable fragment VH and the light chain variable fragment VL gene by the cDNA. The VH and VL was assembled into scFv gene fragment by a 15AA Linker. Constructed the vector of pCANTAB-5E-scFv then transformed it into E.coli TG1, accepted the fusion protein with cpⅢ,forming a scFv phage display library.ELISA was used to select a high affinity scFv antibody after enrichment-panning 3~5 rounds, and ELISA, to get one high affinity and stability scFv-phage. infected the E.coli HB2151 with the scFv-phage to achieve soluble expression of the scFv.