Construction Of Recombinant Antibody Expression System Toward Understanding Factors Required For The Secretion

Biopharmaceuticals are pharmaceutical drugs produced by biological resources.Protein-based pharmaceuticals such as monoclonal antibodies have high specificity and high affinity against target molecules and have low side effects,compared to traditional small molecular compounds.Recombinant immunoglobulin is becoming very important for treating intractable diseases such as cancer and autoimmune diseases.Secreted recombinant immunoglobulins are synthesized in the ER,where they are glycosylated,folded and assembled.Correctly folded immunoglobulins are sorted at the ER,then are transported to the Golgi apparatus,where the glycan is further modified.After the processing,they are secreted to the medium.This project aimed to construct a system to monitor the secretion of recombinant immunoglobulins in mammalian cells.HyHEL10,which is a well characterized antibody against the hen egg lysozyme,was used as a model antibody to express in HEK293 cells and HAP1 cells,which has a human haploid genomic background.A monomeric EGFP and a FLAG-tag fused to the N-terminal of HyHEL10(EGFP-F-IgG)could reflect the levels of the antibody expressing of the cells.The intracellular EGFP-F-IgG was monitored by EGFP fluorescence using flow cytometry,whereas the secreted recombinant EGFP-F-IgG was detected by western blotting and ELISA.In HEK293 cells expressing EGFP-F-IgG,treatment of inhibitor for secretory pathway(brefeldin A)impaired the antibody secretion into medium,while the antibody accumulation level was correspondingly increased inside of the cells.Influence of protein synthesis inhibitor(cycloheximide)led to the suppression of both the antibody synthesis and secretion.The same system was later constructed in a human haploid cell line,HAP1,for genetic screening.Piggy Bac(PB)transposon was used for gene-trapping,whose vectors were inserted into the genome of HAP1 EGFP-F-Ig G at the aim of screening factors related to antibody expression and secretion.PB mutants with a high antibody expression level were isolated by cell sorting,and were then constructed into FLPe resorted cells using recombinase flippase.Flow cytometry and ELISA results showed that three PB mutants(PB C4,PB C11,and PB C13)all had an increase in both antibody expression and secretion,while their corresponding FLPe resorted cells had a similar antibody expressing and secreting capability as the original cell line.The gene-trapped genes by PB transposon were further analyzed by sequencing.The well-constructed HEK293 EGFP-F-IgG system could be used to monitor the secretion of recombinant immunoglobulin in cells.The recombinant antibody expressed in this system can keep its biological ability well,and can react on protein secretion or synthesis inhibitor sensitively.EGFP can be well used for monitoring of antibody expression and secretion.With the advantages of human haploid genomic background of HAP1 cells,PB mutants with a high antibody expression level were screened by gene trapping.Corresponding genes mutated in these mutants would be involved in antibody expression and secretion.