Preparation,Screening And Identification Of Lymphocyte Activation Gene-3 Mutant Library

Lymphocyte activation gene-3(LAG-3)is selectively expressed in activated T cells and natural killer cells.It is a cell surface molecular protein with negative regulatory effects on T cells.LAG-3,as a soluble molecule,activates antigen-presenting cells through MHCII signals,leading to increased antigen-specific T cell responses in vivo.Recent studies have shown that FGL1/LAG-3 is a novel immunosuppressive pathway,by which T cells resume cytotoxic effects and thus limit the tumor growth.In this study,LAG--3 mutated gene was obtained through three rounds of PCR amplification through the site-specific mutation method,and the LAG--3 mutated gene was connected with the vector pCANTAB5E,and then transformed into E.coli TG1 to construct LAG-3 mutant library.Using the solid phase carrier FGL1 protein,the LAG-3 mutant library was screened for 16 positive clones after three rounds of enrichment and phage ELISA.One of the highest positive values was selected for expression and identification.First:the LAG-3 gene and the LAG-3 mutant gene(LAG-3M59)were connected with the vector pCANTAB5E,and were transformed into E.coli TG1.After packaging with the helper phage M13K07,the binding activity of LAG-3M59 and LAG-3 by using FGL1-coated solid phase carrier and ELISA.Second:the LAG-3 gene and the LAG-3M59 gene were connected with the vector pFUSE-hlgG4-Fc2,transformed into Trans-T1 of E.coli,plasmid extraction was performed,endotoxin treatment was conducted,and transfected into eukaryotic cells expressing 293,after purification,LAG-3M59 protein and LAG-3 protein were finally obtained.The binding activity of LAG-3M59 protein and LAG-3 protein by using FGL1-coated solid phase carrier and ELISA.The main results were as follows:(1)the capacity of LAG-3 mutant library is 5×106,after colony identification,the transformation efficiency of mutant library was 65%.(2)The 16 different positive clones screened by Phage ELISA were selected.One clone with the highest OD450nm absorbance value was selected and named LAG-3M59 and identified.(3)Phage ELISA proved that the binding activity of LAG-3M59 and FGL1 was 2 times higher than that of LAG-3;eukaryotic expression of LAG-3M59 protein and LAG-3 protein showed that the binding activity of LAG-3M59 protein and FGL1 was higher than LAG-3 protein 3 times out.