Construction Of Anti-mice CD40 Single Chain Fragment Variable Phage Display Library And Screening A High Affinity CD40 ScFv

Objective: Construct a phage display library of rat anti-mice CD40 single chain fragment variable(Sc Fv) in order to harvest a high affinity CD40 Sc Fv.Methods:(1)The Lewis rats were immunized by injecting mouse recombination CD40 protein to back subcutaneously.(2)The gene of rat anti-CD40 antiboby' Heavy Variable and Light Variable were amplified by reverse transcription PCR( RT-PCR) from total RNA isolated from spleen of the immunized Lewis rats.(3)Sc Fv were constructed by splicing overlap extension PCR( SOE-PCR).(4)The Sc Fv were cloned into phage plasmid p CANTAB5 E by double restriction enzyme to construct recombinant plasmid p CANTAB5E-Sc Fv.(5)The recombinant plasmid p CANTAB5E-Sc Fv was transformed into TG1 E.coli, thus the rat anti-mice CD40 expression library was created.(6)Then, the phage library of rat anti-mice CD40-Sc Fv was built by reinfection of help phage M13KO7.(7)The capacity and diversity of phage display antibody library can be analysized by counting the plaque forming unit of gradientdilution method and selecting the positive clones to sequence.(8)The target antigen of mouse recombination CD40 protein was used to screen the affinity CD40-Scfv through selection cycles and we sequenced the positive clones to analyses the library diversity.Results:(1)The VH?VL gene of mouse CD40 were successfully gained from the immunized Lewis rats.(2)Total Sc Fv gene were gained by SOE-PCR, and gene length was 740 bp.(3)The recombinant plasmid p CANTAB5E-Sc Fv was cut by double restriction enzyme, the result indicated that there were two stripes in 740 bp and 4200 bp. DNA sequencing demonstrated that the variable region of heavy chain(VH) and the variable region of light chain(VL) were assembled into a single chain.(4)The anti-CD40 Sc Fv phage display library were successfully constructed after the TG1 transformed by recombination plasmid p CANTAB5E-Sc Fv was rescued by help phage M13KO7.The size of rat anti-mice CD40 Sc Fv phage display contains 1.2×107pfu/mland the titre of phage is 1012.(5)18 positive clones from the library was selected to sequencing randomly. The results indicated that the alignment and length of insertion sequence were right, which were matched on the basis structure of rat antibody. The mouse CD40-Scfv phage display library had a good diversity.(6)After the two selection cycles under 104 selection stress. The sequence of selected positive clones indicated a trend of convergence.There are some more selection cycles to get a high affinity CD40 Sc Fv.Conclusion:(1)The phage display library of anti-CD40 Sc Fv was constructed successfully.(2)The capacity and diversity of phage display library can afford the penning forreceiving the high binding affinity anti-CD40 Sc Fv in the next procedure.(3)The titre of recombinant phage output of each panning decreased as the screening stress increased,which indicated that the penning strategies of phage display antibody library was appropriate,and lay a foundation for the next experiment.