The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin ...The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.展开更多
基金The Science and Technology Project Foundation of Guangzhou,China(Grant No.201604020106)Natural Science Foundation of Guangdong Province(Grant No.1814050002837)
文摘The primers were designed according to the gene sequence of lipocalin protein family,and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR.The random mutation lipocalin library was constructed using phagemid expression vector.Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells,and the lipocalin secondary library binding to mast cells was obtained.Then the lipocalin secondary library was enriched and screened with FcεRI-αreceptor protein as target molecule,and specific binding phages were eluted.After three rounds of screening,eight recombinant phage clones were randomly selected from elution clones of the third round.ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-αreceptor of mast cells.These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-αreceptor,and also lay the foundation for the development of biological small molecule drugs to treat Ig E associated allergic diseases.
