Affinity maturation of human scFv anti-TSLP in vitro / 中国免疫学杂志

Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.

Selection,soluble expression and identification of full human scFvs against IL-13 / 中国免疫学杂志

Objective:To select specific and neutralizing scFv against IL-13 and soluble expression and identification them.Methods:In our previous study the scFv library were constructed,and here the scFv library was enriched and the positive scFv were screened from the enriched scFv library for three round.The specific positive scFvs with better affinity were ligated with expression vector for expression and identification.Results:After three rounds of enrichment,30%of clones were positive.The two specific scFvs with better affinity and neutralization were selected from almost 500 clones and then ligated with expression vector LZ16 for soluble ex-pression.The expressed scFvs were identified by western blot and biomolecular interaction analysis.Conclusion: The specific scFvs against IL-13 with better affinity and neutralization had been selected successfully.

Clone and soluble fusion expression ofα-HL of Staphylococcus aureus / 中国免疫学杂志

Objective:Expression and purification of the α-HL of Staphylococcus aureus as antigen for making full human anti-α-HL antibody later ,providing of new treatment for Staphylococcus aureus infection.Methods:The total RNA of Staphylococcus aureus was extracted and the cDNA of α-HL was amplified by RT-PCR.The DNA of α-HL and pCold-TF plasmid was digested and ligated by T4 ligase and then transformed into E.coli TOPO 10.The recombinant plasmid α-HL/pCold-TF which verified by sequencing was trans-formed into E.coli BL21 for expression.The expression products was identified by SDS-PAGE and Western blot.Results: The size of amplified cDNA of α-HL was about 900 bp and the expressed soluble fusion protein of α-HL was about 90 kD(including the molecular chaperone in the vector ) after inducing expression for 24 h at 15℃.The Western blot results showed that the expressed protein was the fusion protein of α-HL.The purified α-HL was injected into BABL/c mice for making antiserum.The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10 000 times.Conclusion: The α-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of α-HL was successfully expressed.

Clone,expression and identification of human recombinant IL-4 / 重庆医学

Objective Construction of human IL‐4 recombinant expression vector and then conduct the expression ,purification and identification of human recombinant IL‐4 .Methods the open reading frame of IL‐4 was amplified by nest PCR with total RNA from PBMC of healthy volunteer .And then the amplified IL‐4 was inserted into pET101/D‐TOPO ,transformed into BL21 ,ex‐pressed ,purified and indentified .Results The size of amplified open reading frame of IL‐4 was about 460 bp and the sequence was correct .After transformed into BL21 ,the IL‐4 clone with higher expression level was selected by selection of different clones insert‐ed with IL‐4 and the size of expressed ,purified IL‐4 was about 28 × 103 .Western blot results showed that the size of single band was identical with the expected protein .Conclusion Human IL‐4 recombinant protein was got successfully .

Selection and identification of full human scFv against TSLP / 中国免疫学杂志

Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.

Construction and identification of non-immunized human phage display library / 中国免疫学杂志

Objective:To develop non-immunized human phage display library.Methods:The total RNA of lymphocyte cells from peripheral blood of healthy voluntee was isolated and cDNA was synthesized,and the genes of heavy variable chain (VH) and light variable chain (V? and V?) were amplified by direct PCR and half-nested PCR.By overlapping extension PCR,the genes of VH and VL (V? and V?) were linked.The linked genes of single chain Fv fragment (scFv) were ligated with the vector pCANTAB-5E and then cloned into TG1 for the scFv library construction.Results:By direct PCR and half-nested PCR,42 VH fragments,16 V? and 18 V? fragments were obtained.The size of linked scFv library genes was 750 bp and the volume of constructed scFv library was 1.35?108.The results of BstN Ⅰ analysis of scFv genes from the phage library showed that fingerprint map of the selected scFvs was different.Conclusion:The developed phage library is diversity and can be used for selecting humanized scFv.