基因重组IgG1 κ抗-P质粒构建及其在HEK293T细胞的表达OA

Objective To construct gene recombinant expression plasmids of human anti-P antibody with IgG1 and kap-pa chain based on the human hybridoma cell line.Methods Starting from the specific RT-PCR products encoding the var-iable regions of the IgH chain and IgL chain of the anti-P monoclonal cell line,appropriate restriction enzyme digestion sites were introduced at both ends of the VH and VL fragments through nested PCR.The plasmids carrying the antibody constant region and the nested PCR products of VH and VL were ligated by the action of T4 ligase and subsequently transferred into competent E.coli DH5ɑ,and positive clones were selected in the antibiotic resistant LB medium.After sequence confirma-tion,recombinant plasmids DNA were transfected into HEK293T cells,and the recombinant antibody obtained in the culture supernatant.The characteristics of recombinant expression antibodies were determined by using rapid antibody isotying kit,serological agglutination tests and flow cytometry.Results Recombinant gene expression vectors,pFUSEss-CHIg-hG1+VH,pFUSE2ss-CLIg-hκ+VL,were successfully constructed.Human IgG1 kappa light chain antibodies were detected in the supernatant of HEK293T cells transient transfected with recombinant plasmids.After the supernatant was ultra-filtered and concentrated,it could cause agglutination reactions with P antigen-positive red blood cells.The mean fluorescence intensity(MFI)of the reaction between recombinant antibodies and antigen-positive red blood cells in flow cytometry experiments was higher than that of antigen-negative red blood cells.Conclusion The experimental study on the conversion of red blood group antibody types by genetic engineering technology represents a beneficial exploration towards establishing a feasible technical route for the development of genetic recombination and modification of antibodies reagent.