Construction and Identification of a Single Chain Fv Phage Display Library Against Human Umbilical Cord Mesenchymal Stem Cell / 中国生物工程杂志

ABSTRACT

Objective:To construct and identify a ScFv phage display library against human umbilical cord mesenchymal stem cells.Methods: BALB/c mice were immunized with cultured UC-MSCs.After the third immunization,the total RNA was extracted from the spleen cells of the immunized BALB/c mice and purified by affinity chromatography with mRNA Purification Kit.The heavy-chain and light-chain variable region genes(VH and VL) were amplified by PCR using relevant primers.PCR products of VH and VL genes were cloned into the phagemid vector pSEX81 and electroporated into the XL1-Blue strain of E.coli.The ScFv phage display library against human umbilical cord mesenchymal stem cells was constructed and the capacity of library was measured.The library was panned by three cycles and screened with purified UC-MSCs.The percentage of clones containing a full-length scFv-encoding insert and their diversity was determined for unselected and selected libraries.Results: The amplified fragments of VH and VL genes by RT-PCR were about 399bp and 357bp,respectively.VH and VL genes were all successfully cloned into the phagemid vector pSEX81,which were confirmed by the amplication of 786bp full-length scFv fragments by PCR.The ScFv phage display library had a capacity of approximately 2?107 cfu.After three cycles of panning,PCR of plasmid DNA prepared from 15 individual phage clones showed that the recombination rate increased from 93% to 100%.BstN1 fingerprinting of insert DNA showed that the diversity of clones decreased with increasing rounds of selection.After three rounds of selection,3 clones showed an identical restriction enzyme pattern.There was a 330-fold enrichment of library phage after 2 rounds of selection and after 3 rounds,a further 8-fold enrichment of library phage was obtained.Conclusion: The ScFv phage display library against human umbilical cord mesenchymal stem cells was successfully constructed.It can be used for succeeding screening of specific antibody against human umbilical cord mesenchymal stem cells and further studying of the cell surface molecules of mesenchymal stem cells.