Construction Of Camelidae Natural Nanobody Phage Dispaly Library And Screening For Anti-CD19 Nanobody And Verification

Objective In order to construct and verify the natural nanobody phage display library for screening of nanobodies against various antigen molecules.Using CD19 as the antigen to screen and obtain VHH of the camel targeting CD19.Then to express and purify the nanobody,and verify their antibody binding ability.Methods In order to obtain c DNA,extracting the total RNA from spleen of Bactrian camel,and using the Oligo DT to perform reverse transcription from the total RNA,and its heavy chain variable region gene fragment was obtained by nested PCR.The p CANTABA5e phagemid was selected as a phage display vector,the vector and target fragment were digested with endonuclease.Then to ligate p CANTABA5e phagemid and the target fragment to construct a phage display vector.Electrotransformation this vector into TG1 E.coli and KM13 helper phage rescue to construct a phage display library,and analyze and identify the capacity and diversity of nanobody phage display library.With the CD19 as the antigen,and the biotinylated CD19 antigen was inoculate with streptavidin magnetic beads in the liquid phase,and the magnetic beads were collected by a magnetic stand.After washing the magnetic beads repeatedly,the phages attached to the magnetic beads were eluted.The phages obtained from the elution was used to infect the TG1 E.coli in the logarithmic growth phase,and the phage was collected after amplification and collected for the next round of screening.The screening process was repeated three times to enrichment the monoclonal phage with expressing anti-CD19 nanobody on the surface.Phage-ELISA was used to identify of monoclonals after screening,monoclonal clones with higher positive values were selected to sequencing,analyze and verify.Anti-CD19 Nanobody with excellent analytical data was selected to expression and purification.The target Nanobody sequence was linked to the human Ig G Fc segment and ligated between the Nde I and Xba I sites of the vector p CZN1 to obtain a recombinant plasmid.The recombinant expression plasmid was transferred into Top10 bacteria for the expression and purification of nanobody.The purified anti-CD19 nanobody were subsequently verified by SDS-PAGE and Western Blot.The target protein was verified by antigen binding,with CD19 as antigen,target protein as primary antibody,and anti-human Ig G/HRP as secondary antibody for Weatern Blot validation.At the same time,CD19+NALM-1 cells were cultured,and the whole protein was extracted as antigen for Western Blot to verify the specificity of the recombinant protein binding to CD19 antigen.Results 1.The constructed natural phage nanobody display library has great diversity,and its fragment insertion rate is about 95%.The amino acid homology of 20 randomly selected clones is 65.85%,and the titer of the display library after rescue by phage is1.0x10¹³CFU/ml.The evolutionary tree of the MEGA system was constructed to show that the displayed library had a great diversity.2.After four rounds of phage display library panning with CD19 as the target antigen,Phage-ELISA was performed on the phage monoclonals obtained after panning,and the monoclonals with higher positive values were selected to sequencing.BLAST analysis of the sequencing results showed that they were camel-derived nanobodies.The results of multiple sequence alignment showed that obtained three anti-CD19 nanobodies,named:CD19-01,CD19-02 and CD19-03.And the three sequences showed great hydrophilicity,and some hydrophobic amino acids in the CDR1 and FR2 regions were mutated to hydrophilic amino acids.3.The CD19 sequence was selected for expression and validation,and ligated to the human Ig G Fc segment,and construct a recombinant expression vector with p CZN1plasmid.Transferred this vector into the Top10 bacteria,and useing IPTG induced the expression.According to the actual condition of SDS-PAGE of the expressed product,the protein was expressed as inclusion bodies.After the protein renaturation,the protein was purified by affinity purification on a Ni column.The anti-His tag was used to perform Weatern Blot verification on the recombinant protein,and the results indicated that the Mr of the target protein was about 40 Kda.Subsequently,the target protein was verified for antigen binding,and the results showed that the target antibody could specifically bind to the antigen CD19.The binding affinity of Nanobody to human CD19 by surface plasmon resonance technology is 0.29μM.Conclusion Successfully constructed a camelidae natural nanobody phage display library with high titer and good diversity.Three anti-CD19 nanobody sequences were obtained by panning with CD19 as antigen.The expression and validation of the nanobodies was consistent with the characteristics of anti-CD19 nanobody.Provide technical support for the research of diagnostic kits and antibody drugs targeting CD19,and provide theoretical basis and support for the preparation of CAR-T cells targeting CD19.