Preparation And Identification Of A Single Chain Fragment Variable (scfv) Against Needle Subunit (vpl694)

Vibrio parahaemolyticus, a halophilic game-negative bacterium, is a humanpathogen that naturally inhabits marine and estuarine environments. Infection with V.parahaemolyticus may not only lead to certain diseases in aquatic species but alsolead the development of acute gastroenteritis characterized by diarrhea, nausea,vomiting and so on. However, the pathogenic mechanism of V. parahaemolyticus isnot entirely clear, and there is no preventive measure against it and no totally effectivetreatment. Recently, Type III secretion systems (T3SS) was found to be associatedclosely with virulence, and the needle plays an important role in this systems. In thisstudy, A phage-scFv library against needle was constructed by Overlap-PCR and aspecial scFv against needle was obtained by phage display technology, namedanti-vp1694-scFv. Subsequently, a capable purification scFv was generated byco-expression method. Our results can provide a reference for the prevention andtherapy of V. parahaemolyticus induced by T3SS, and might used for the furtherreseach of pathogenic mechanism of T3SS in V. parahaemolyticus.Firstly, the needle gene (vp1694) was obtained from NCBI, which was cloned totwo recombinant vectors, so-called pGEX-6p-1-vp1694and pET32a(+)-vp1694.Accordingly, GST-vp1694and TRX-vp1694fusion proteins were purifiedrespectively. After the determination of protein concentrations, GST-vp1694proteinwas used to immune of BALB/c mouse, and TRX-vp1694protein was used to thedetection of the serum titer assay in ELISA. GST-vp1694concentration wasapproximately0.237mg/mL and TRX-vp1694concentration was0.942mg/mL.After3times immunizaction, the serum titer could be up to1/16000detected byELISA. Then the total RNA of spleen has been extracted. After RT-PCR, the cDNAwere obtained, as the template, and cDNA was used to amplify the VHand VLgene.Then, the entire scFv gene containing VH-Linker-VLsequences was amplified byOverlap-PCR, and was subcloned into pCANTAB5E vector to constructedphage-scFv antibody library. Finally, the storage capacity of the phage-scFv antibodylibrary was up to1.4×107cfu/mL. After six rounds of screening by phage display technology, two monoclonalstrains were obtained, named FA7and FE9respectively. By sequence analysis, theamino acid sequences are silimar, and its composed by729base pairs. ELISA wascarried out to detect the specificity of anti-vp1694-scFv. It shown that the reactionbetween anti-vp1694and vp1694was significant (P<0.01), but the reaction betweenanti-vp1694and TDH, TLH, VopQ and HrpA was weak, which indicated thatanti-vp1694-scFv has a good antibody specificity.Finally, the scFv gene was amplified from pCANTAB5E, and was subcloned intopACYC–Duet–1-Skp co-expression vector contained Skp chaperone protein, whichcan help the soluble expression of scFv. Ni2+-NTA meathod was used to purify thescFv protein. Finally its concentration was up to0.102mg/mL by BCA kit. After thedetermination of antibody affinity, the Kaff of anti-vp1694-scFv was approximate1.07×108.Besides, the IMGT/V-QUEST database was carried out to analyze CDR regionand hemologous of anti-vp1694-scFv, its found that VHgene classified IGHV5,IGHJ2and IGHD2while VLgene classified IGKV4and IGKJ2. Finally the analog ofamind acid was conducted by IMGT/Collier-de-Perles datebase, which might used forthe further reform of anti-vp1694-scFv.