Effect Of Intron And PolyA On Transgene Expression Of CHO Cell Expression System

BackgroundCurrently,nearly 70% of the production of therapeutic recombinant proteins(including recombinant antibodies)worldwide are produced by the expression system of Chinese hamster ovary(CHO)cells.There are many factors influencing the efficient and stable expression of recombinant protein in CHO cells,among which the expression vector is part of the most important.Because of the random integration of the target gene and the frequent occurrence of gene silencing and unstable expression,the development of large-scale industrial production is restricted.Therefore,the construction of efficient and stable expression vector has become a hot topic of domestic and foreign scholars.ObjectiveTo study the effects of different intron elements and polyA elements on the expression level and stability of CHO cells were studied and the mechanism was discussed.Methods1.According to intron sequences hCMV intron A(hCMVI),TPL intron(TPLI),SV40 intron(SVI)and CHEF1 gene intron1(CHEFI)reported in the literature,PCR amplification or artificial synthesis of intron fragments replaced intron IVS in the original expression vector pIRES-EGFP to construct expression vectors containing different introns.2.According to the reported polyA sequences(BGH polyA,Mutation BGH polyA,Synthetic polyA,HSV TK polyA,SV40 late polyadenylation signal),PCR amplification or artificial synthesis of polyA fragments replaced the polyA in the original expression vector pIRES-EGFP,and the expression vectors containing different polyA fragments were constructed.3.CHO-S cells were transformed with the constructed vector using lip2000 transfection reagent,and the transfection was observed under a fluorescence microscope 48 h later,and its transient expression and transfection efficiency were detected by flow cytometry.4.The stable expression of eGFP was detected by flow cytometry of clone strains with stable expression under screening pressure,and the transgenic copy number and mRNA expression were detected by qPCR and RT-qPCR to screen the high-expression vector.5.The reporter gene eGFP on the high-expression vector screened by adam antibody gene substitution was used to construct the expression vector containing the target gene to transact CHO-S cells and detect the expression of adam antibody by Western Blot.Results1.it was proved by enzyme digestion and sequencing that the recombinant vector had been successfully constructed.2.Transient expression analysis showed that compared with the control vector,the SV40 intron in the intron could improve the transfection efficiency and transient expression(P<0.05),while BGH polyA and Syntion polyA could significantly improve the transfection efficiency and transient expression compared with the control vector(P<0.05).3.Steady expression analysis showed that both SV40 intron and hCMV intron could increase the expression of CHO cells(P<0.05),while for ploy A,synthetic polyA and SV40 late polyA,the stable expression of CHO cells could be significantly increased(P < 0.05).4.qPCR indicated that the level of transgenic expression was independent of gene copy number.MRNA expression of eGFP was detected by RT-qPCR,and the results showed that the expression level of mRNA was not positively correlated with the expression level of the target gene.5.Western blot results showed that adam antibody could also be highlyexpressed in the screened high expression vector.Conclusion1.SVI and hCMVI in intron can significantly increase the expression of transgene in CHO cells.2.Synthetic and SV40 late in polyA can significantly increase the expression of transgene in CHO cell3.The screened high expression vector can also improve the expression of adam antibody.