| The low transfection efficiency by gene delivery systems has limited the therapeutic utility of gene transfer.Though vectors based on virus can deliver relatively high level of gene expression,the location it inserts or integrates into the chromosome is random and it may result in the potential danger of insertion mutation and cell malignant transformation.The non-viral gene delivery systems,with low immunogenicity and some advantages in transient expression of exogenous gene,safe and reliable,suitable for mass production,have been widely utilized in various types of development for DNA vaccine.Nevertheless,some disadvantages are still existing such as low transduction efficiency in vivo,poor targeting and short-time expression of desired gene.Given the better biological safety of non-virus gene delivery systems,more and more attention has been given by people to enhance the exogenous gene expression and then extend the time of gene therapy.In addition,the expression technology of recombinant proteins,as a valuable tool in studying the structures and functions for some proteins or screening of targeted drugs,plays a crucial role in the field of basic science and medicine.So far,a variety of prokaryotic and eukaryotic expression systems for recombinant proteins have been exploited by people.It is convenient,cheap and short cycle for the prokaryotic expression systems,nonetheless,the lacking ability to protein folding and glycosylation modification has limited their applications.The mammalian expression system,as a typical eukaryotic expression system,is mainly exploited for the preparation of secretory recombinant proteins and superior to other expression systems in protein folding and disulfide bond formation.The expressed proteins are the closest to natural,which can be benefitical to studying structures and functions for some proteins.However,some disadvantages such as lower productivity and higher cultivation cost are also existing.Therefore,a simple and efficient transient expression system is expected to improve the production efficiency of recombinant proteins.Additionally,while exogenous DNA or RNA presented by recombinant saccharomyces cerevisiae can produce specific antibodies and killer T cells,the specific immune response may be slower and the dose smaller.The Gal4/UAS system is a biochemical method utilized to study gene expression andfunction in organisms.In this study,the self-activating Gal4/UAS expression cassette was added in the CMV-based expression vector to increase gene expression.Firstly,two upstream activating sequences(2UAS),three DNA activating domains including VP64,Gal4 AD and VP64-p65-Rta were compared,and pRS426-2UAS-CMV-Luciferase-T2A-NLS-Gal4BDVP64-NLS exhibited the most Luciferase expression(> 16 folds).Secondly,2UAS was replaced by 4UAS to build the vector pRS426-4UAS-CMV-Luciferase-T2A-NLS-Gal4BDVP64-NLS,and this vector could enhance the report gene expression of Luciferase by nearly23-fold higher than only CMV-based expression cassette.Thirdly,the stability of enhancd gene expression levels in HEK293 T cells was detected and the expression level of Luciferase initiated by the vector pRS426-4UAS-CMV-Luciferase-T2A-NLS-Gal4BD-VP64-NLS maintained higher from 6th to 10 th day post transfection in HEK293 T cells and at about 8th reached the highest compared with that activated by the CMV-based expression cassette.In addition,the enhancement effect was verified in CHO cells which was often employed specially to produce recombinant proteins(> 8 folds).Taken together,with Gal4/UAS expression cassette in the CMV-based vectors,it can produce an autocatalytic,exponential increase in gene expression.The Gal4/UAS expression cassette,as a novel enhancement system,is expected to improve the production of recombinant proteins and become a powerful tool in the non-viral gene mediated pathway,and play a crucial role in prevention and immunization of diseases by the DNA vaccine presented through recombinant yeast. |
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