Construction Of A Human ScFv Phage Display Library And Selection Of Antibody To Human TNF-α

Antibody plays an important role not only in the immune system, but also inclinical diagnosis, pharmaceutical and medical researchs. Antibody phage displaytechnology is one of the favourite tools for generating humanized therapeuticantibody. TNF-α is a primary proinflammatory cytokine and is considered to be apotential mediator involved in variety of human diseases, including rheumatoidarthritis （RA）. In this study, a phage antibody display library was constructed, andfrom this library we screened a scFv with the potential ability to inhibit the cytotoxicactivity of TNF-α on L929cells.In this study, all the primers, used to amplify the gene encoding humanantibody variable regions, were designed according to the publication informationand modified. A series of PCR amplification ractions were performed with42primers and total RNA which was isolated from lymphocytes of200donors. Threerounds of PCR reactions were performed to isolate and assemble the VH genes.Firstly, four pairs of primers were used to amplify the CDR1, CDR2, FR3andCDR3, respectively. Secondly, OE-PCR was performed respectively to join CDR1and CDR2, FR3and CDR3together. Finally, the whole VH was generated fromOE-PCR, and the VL was obtained by PCR simultaneously. Phagemid vectorpCANTAB5E and scFv were digested with restriction enzyme Sfi I and Not I, andligated with T4DNA ligation enzyme. The newly constructed vector was named aspCANTAB5E-scFv, and transformated into competent cell E.coli TG1strain. Thenthe transformated cells were infected by the helper phage M13KO7, the recombinantlibrary with scFv was displayed on the surface of filamentous phage. Gradientdilution method showed that the library contains2.5×10⁷individual clones and10¹²PFU. The results of DNA sequencing demonstrated that the gene inserted the phagewith a normal framework, and the VH and VL genes connected with a flexibilypeptide. Increased binding phages against five proteins, TNF-α, HSA, NS1-GTS, GP5and OVA in the panning process indicated that this phage library can be effectively enriched bythe five proteins.Phage ELISA was performed to select and isolate high affinity recombinantphage clones and four monoclone phages were picked from87random picked phageclones. Recombinant proteins scFv were expressed in E.coli BL21（DE3） strain with prokaryotic expression vector pET-28a, and induced with IPTG in2×YT medium.SDS-PAGE showed that recombinant proteins scFv were expressed with40%tropinaand obtained by His tag purification tube with more than90%purity. MTT detection resultsshowed that the ED₅₀ of TNF-α on L929cells was0.01ng/mL with the synergy of10μg/mLdactinomycin. Gradient diluted recombinant protein scFv effect on the0.01ng/mL of TNF-αshowed that the recombintant protein B11and A24with a IC₅₀ of70μg/mL and100μg/mL,respectively.In conclusion, we constructed, indentified a human scFv phage display library, andsuccessfully selected2human scFv with the potency of inhibition of TNF-α. This work laidthe foundation for the development of anti-TNF-α antibody drugs.