Construction Of Human Phage Display Single Chain Fv Library And Screening Of Anti-IGF-1/IGF-2 Single Chain Fv

BackgroundInsulin-like growth factor-1?IGF-1?and IGF-2 are highly expressed in various types of tumors,and are closely related to the poor prognosis of patients.Therefore,they are ideal targets for molecular targeted drug development.A single-chain antibody?scFv?is composed of heavy chain variable region?VH?and light chain variable region?VL?which are linked by a small flexible peptide.It has the advantages of small molecular weight,strong penetration,low immunogenicity and easy to construct,etc.Phage display technology is to insert the gene fragment of antibody protein into the gene encoding the coat protein of the phage,so that the antibody and coat protein can be fused and expressed on the phage surface.After several rounds of screening of the phage antibody library with specific antigen,a single chain antibody for specific antigen can be obtained.In this study,a human phage display single chain antibody library with large capacity was constructed firstly,and then a novel specific anti-IGF-1/IGF-2 scFv was obtained by screening this library.ObjectiveIn this study,a human large capacity phage display single chain antibody library was constructed,from which a scFv with high affinity to IGF-1 and IGF-2 and relatively low affinity to insulin was screened.It not only provided the necessary basis for the next step to study the anti-tumor activity in vitro and in vivo of the scFv,but also was a new exploration for the development of targeted drugs for IGF signaling system.Methods1.The peripheral blood samples of 50 healthy people or patients were collected,and the lymphocytes were isolated and total RNA was extracted.The VH and VL gene of the antibody were amplified by reverse transcription polymerase chain reaction?RT-PCR?,and then the scFv gene was constructed by linking the VH with VL by a?G4S?3 linker using the overlapping extension splicing polymerase chain reaction?SOE-PCR?.The scFv gene?VH-linker-VL?was inserted into the phage vector pCANTAB-5E,and then the recombinant vector was electrotransformed into E.coli TG1 competent cells to construct a primary phage display scFv library.2.The plasmids of 15 randomly selected clones from the primary library were extracted,and the scFv genes were amplified,enzyme-digested?Sfi I and Not I?and sequenced.The insertion efficiency,the diversity and the homology to immunoglobins of the library were analyzed.3.Using IGF-1 and IGF-2 as antigens,a secondary scFv library with high affinity for IGF-1 and IGF-2 was screened through seven rounds of binding,washing,elution and amplification process.4.93 clones were randomly selected from the secondary antibody library,and the recombinant phage clones with the highest affinity with IGF-1and IGF-2 were screened by ELISA.The DNA sequence of single chain antibody was obtained by sequencing,and blast analysis was carried out in NCBI database.5.Based on the sequencing results,the gene of scFv was synthesized after codon optimization,and it was inserted into the expression vector pET30a under an IPTG inducible promoter to obtain the recombinant expression vector pET30a-anti-IGF-1/IGF-2-scFv.6.The recombinant vector pET30a-anti-IGF-1/IGF-2-scFv was transformed into E.coli BL21?DE3?star^TM cells and then the IPTG was added into the LB-medium to induce the expression of the scFv protein.SDS-PAGE and Western blot were used to analyze the location of scFv protein in E.coli.7.The factors affecting protein expression were optimized,including initial cell density the culture temperature,the induction time,and the IPTG concentration.8.The scFv protein was purified by Ni²⁺affinity chromatography under denaturing condition and the denatured scFv protein was refolded by stepwise dialysis to obtain the active soluble protein.The concentration of purified scFv protein was determined by BCA,and the purity was detected by SDS-PAGE and Western blot.9.The binding affinity anti-IGF-1/IGF-2 scFv with its corresponding antigens,e.g.IGF-1,IGF-2 and insulin,was detected by ELISA.Results1.The VH and VL genes were amplified by RT-PCR from the total RNA of peripheral blood lymphocytes of 50 healthy people or patients.The VH and VL genes were 340bp and325bp,respectively.The scFv gene,750bp in length,was constructed by linking the VH with VL by a?G4S?3 linker using the overlapping extension splicing polymerase chain reaction?SOE-PCR?.2.The scFv gene?VH-linker-VL?was inserted into the phage vector pCANTAB-5E,and then the recombinant vector was electrotransformed into E.coli TG1 competent cells.A primary phage display scFv library with a capacity of 4.50×10⁷?kappa chain?and 3.49×10⁹?lamda chain?was constructed.The results of sequence alignment showed that there was a high diversity among the clones,and BLAST alignment in NCBI database showed that the scFv library had a high homology with human immunoglobulins.3.The primary antibody library was screened with IGF-1 as antigen for three rounds,and then with IGF-2 as antigen for four rounds.The secondary single chain antibody library with high affinity with IGF-1 and IGF-2 was obtained.4.93 clones randomly selected from the secondary antibody library were screened by ELISA.It was found that clone 9 had the highest affinity with IGF-1 and IGF-2 and relatively low affinity with insulin.The sequencing results showed that the scFv from clone 9 was651bp long?the length of VH,VL and linker was 384bp,222bp and 45b,respectively?.The VH and kappa VL each contains 3 complementarity determining region,CDR)as analyzed by Igblast.5.The codon-optimized pET30a-anti-IGF-1/IGF-2-scFv expression plasmid gene was synthesized.The location analysis from SDS-PAGE and Western blot experiments revealed that scFv protein was in the cytoplasm of E.coli and in the form of inclusion body with a molecular weight about 27kDa.6.The optimal expression conditions of scFv protein in E.coli were,culture temperature:37?,initial cell density:OD600=1.2,IPTG concentration:0.6mmol/l and induction time:8h.7.The scFv protein was purified by Ni²⁺affinity chromatography under denaturing condition and the purity was over 90%as dertermined by SDS-PAGE.After that,the denatured protein was refolded by the step-by-step dialysis method,which gradually reduced the urea concentration,and the active soluble protein was obtained.The yield of scFv protein was 59.53mg per liter fermentation broth.8.The ELISA results revealed that the anti-IGF-1/IGF-2 scFv had strong affinity to IGF-1 and IGF-2,with the Kd value of 7.387?g/ml?0.274?mol/L?and 9.437?g/ml?0.35?mol/L?,respectively.It showed a relatively low affinity to insulin with a Kd value of24.47?g/ml?0.906?mol/L?.Conclusions1.A phage display scFv library was successfully constructed,which has the characteristics of large capacity,high diversity and human resource.It provides a good platform for the screening and development of various scFvs.2.A specific scFv against IGF-1and IGF-2 was obtained by panning the scFv library.3.After expression in E.Coli,isolation the inculsion body,purification by affinity chromatography,and refolding by stepwise dialysis,the anti-IGF-1/IGF-2 scFv protein was successfully obtained.4.The scFv showed a high affinity to IGF-1 and IGF-2,but relative low affinity to insulin.