High Secretive Expression Of Foreign Protein In Mammalian Cells Mediated By Recombinant Adenovirus

Genes are carriers for storage, replication, and expression of biological information in living organism, when proteins are executor and exemplification of life activities. It is not enough for that research in life science only stays in the nucleic acid level, especially in post-genomic era. The primary structure of the protein, namely the numbers, types and order of amino acids, which is determined by the nucleic acid sequence of the genes, is basic of structure and function of proteins. But the structure of proteins is so complex and irregular that there is not still reliable theory to predict structure and function of proteins currently. For example, how proteins fold to form a three-dimensional structure from its amino acid sequence remains still unknown. Since if we want to study a gene, sufficient amount of the protein must be obtained firstly, making use of expression system of recombinant proteins is vey helpful for follow-up study of proteins. Therefore protein is raw material of the scientific study, at the same time expression system of recombinant protein booster research of life science.Recombinant protein expression system contains E. coli expression system, yeast expression systems, insect cell expression system and mammalian cell expression systems, in which each system has its own characteristics. It is E. coli expression system that is widely used at present. Characteristics of each expression system of recombinant protein are described as follows:E.coli expression system of recombinant protein shows its advantage in certain aspects, such as the clear genetic background, easy operation, low production cost and high expression of recombinant protein. Whereas it has a fatal flaw in lack of protein modifications, such as glycosylation functions, suffering in that it is unable to produce foreign proteins with complex structure. Most of the recombinant proteins in E.coli expression system are expressed in form of insoluble inclusion bodies, which need to be refolded in vitro to restore activity of recombinant proteins.Yeast expression system of recombinant protein features both in prokaryotes and eukaryotes, having the advantage in rapid growth, correct modification for recombinant protein and high yield of recombinant protein. But, because of codon bias, it has specific requirement for exogenous gene sequences and still can not modify foreign proteins with very complex structure.Insect cell expression system shows its advantage in high yield of recombinant proteins and has the relatively perfect post-translational modification. Nevertheless, it is worth to note that oligosaccharide chains expressed in mammalian cell expression and insect cell expression system are not identical, and the latter excites deficiency in recognization and cleavage for many protein precursorThanks to perfect glycosylation and proper fold mechanism for foreign proteins, mammalian cell expression system can provide ideal post-translational process and modification for recombinant proteins and then lead them out to extracellular. Currently in mammalian cell expression system there is problem at low level of expression, complex mechanisms of regulation for gene expression, difficulty for downstream purification.When we select appropriate expression system to attain efficient expression of target proteins, complexity of target protein, test requirement and technology should be considered. Although the expression is at low level, mammalian cell expression system could properly express complex recombinant proteins, whose structure is infinitely closed to natural. For example, antigen protein expressed by mammalian cells has good immunogenicity and thus boost immune immunological diagnostic accuracy. Therefore, mammalian cell expression system is mainstream at present. There are two ways to introduce foreign genes into cells:one way is mediated by viral infection such as adenovirus, lentivirus, adeno-associated virus, retrovirus.; another way is to by means of chemical method (for example, liposomes, phosphate calcium, PEI) or physical method (for example, microinjection, electroporation).Adenovirus is a non-enveloped double stranded DNA virus, with the genome of approximately25-45kb that can theoretically encode22-40genes, packaged by the regular icosahedral capsid, with the diameter of80-110nm. Adenoviral vector systems are widely used to express human proteins and non-human proteins, showing advantage at wide range for host cells, low pathogenicity for human, expression in proliferation cells and non-proliferation cells, no risk for gene mutation and cancer. While more exogenous genes could be encoded by recombinant adenovirus and recombinant adenovirus can efficiently amplify in particular cells, high titer (1012PFU/ml) for recombinant adenovirus can be easily obtained. That makes it very suitable for gene therapy in vivo and vaccinationEfficient eukaryotic expression system mainly consists of two parts: efficient secretion expression system and host cells. Efficient adenoviral secreted expression vector is constructed to guide recombinant proteins to medium, that is beneficial for purification of recombinant proteins. The study was conducted to explore in two aspects for exogenous gene expression in animal cells, such as replacement of signal peptides and serum-free culture: Replace of signal peptide to enhance secretion expression of human coagulation factor FVⅡ. Although research has been going on for decades, but expression of human coagulation factor FVⅡ has not significantly improved. The study has used signal peptides of Kappa light chain of mouse antibody(IgK), human interferon alpha(IFN-a), human interferon gamma(IFN-γ), bone morphogenetic protein6in mice (BMP6) and tissue plasminogen activator (tPA) to replace human coagulation factor FVⅡ. We have constructed pAdTrack-CMV-signal peptide-FVⅡ vector and transfected those into293T cells. We have detected protein FVⅡ in medium and protein GPF in cells by Western Blot, then compared the former and the latte to find differences in secretion expression of human coagulation factor FVⅡ due to replacement of signal peptides. The results showed that replacement of signal peptides of Kappa light chain of mouse antibody(IgK) or bone morphogenetic protein6in mice (BMP6) can improve secretion expression of human coagulation factor FVⅡ with3fold increase.As an important part of downstream in genetic engineering, animal cell culture plays an important role in production and scientific research. And cell culture medium is important for animal cell culture. Generally cell proliferation requires regulation from a variety of factors contained in serum. However, in serum there is some defect, for example, quality difference between batches, possibility of mycoplasma contamination, especially complex components that could increase the difficulty of purification. Currently research and development of serum-free medium based on cells and production have been an important topic in cell engineering. In this study, recombinant adenovirus Ad-GFP has been amplified in293A cell and purified by cesium chloride density gradient centrifugation. And the titer of recombinant adenovirus Ad-GFP has been determined by detection of GFP. After mammalian cells (CHO cells, LO2cells, PC3cells) reached95%confluence in serum-free culture conditions, mediums were switched to serum free medium (high glucose DMEM, RMPI1640,293Serum-Free Style), complete medium was used as a control at the same time. Then recombinant adenovirus Ad-GFP was use to infect cells at different MOI. Expression of GFP has been detected at24h,48h,72h, after infection. The result shows that expression of GFP in CHO cells, LO2cells, and PC3cells, that are maintained in high glucose DMEM do not differ significantly from those maintained in complete medium. That imply that animal cells infected by adenovirus is maintained in high glucose DMEM to express recombinant proteins may be an optional strategy.