| Developed in the 1970s, Gene Engineering refers to construct a new genetic recombination by insert different source of foreign gene into a special virus vector or plasmid in vitro, then the vector is transfected into a suitable host cell, in which the foreign gene can maintain stable proliferation and translate the functional protein.The most important thing of Gene Engineering is to produce fuctional protein with significant research value, so far, Gene Engineering has been widely used to produce exogenous protein in various fields included health care, scientific research and industry. Recombinant protein expression system mainly contains E. coli expression system, yeast expression system, insect cell expression system and mammalian cell expression systems; each has its ownadvantages and disadvantages.Bacterial system is the oldest and most widely used expression system, with its advantage of the clear genetic background, easy operation, low production cost and high expression of recombinant protein. However, it also has disadvantage such as lack of protein modifications, which makes the protein inactive and expressing in form of insoluble inclusion bodies.Yeast expression system shows its advantages in rapid growth, high yield of recombinant protein and correct post-translational modification. However, the protein products in yeast are inhomogeneous, with degradation and incomplete signal peptide processing, and it only have part of post-translational modifications, which means it cannot modify foreign proteins with very complex structure..Insect cell expression system is being widely used for routine production of recombinant proteins, with its advantage in high yield of recombinant proteins and the relatively perfect post-translational modification. However, insect cell line-based platforms cannot correctly recapitμlate complex mammalian N-glycan, which lead the different glycosylation of oligosaccharides, campared to the mammalian cells. Other disadvantages include fussy operation and high cost to cμlture.Mammalian cell line-based platform is expected to be an ideal expression system for recombinant protein production, due to its perfect glycosylation modification and folding mechanism. The activity of recombinant proteins in mammalian cells are much closer to native protein, compared to those produced in other systems. But it also has some shortcomings, such as low level of expression, complex regμlation mechanisms for gene expression, tedious, time consuming (often taking months), and costly.When we select an appropriate expression system to attain efficient expression of target proteins, complexity of target protein, technical conditions and experimental requirement should be considered. So far, mammalian cell expression systems become the mainstream in recombinant protein production due to its outstanding characteristics. Although the expression level is low, mammalian cell expression system coμld properly express complex recombinant proteins, whose structure and activity are infinitely closed to natural. The key to the success of mammalian cell line-based protein production is to select a stable and high expressing cell clone. There are mainly two ways to tansform foreign genes into cells one is transient transfection mediated by chemical method (for example, liposomes, phosphate calcium, PEI) or physical method (for example, microinjection, and electroporation); the other is viral vector mediate gene transfection, such as adenovirus, lentivirus, adeno-associated virus and retrovirus. Transient transfection is often inefficient, costly and unstable, and foreign genes could be lost during cell division so it only express for a short time. In comparison, lentiviral vectors have some advantages over the other vectors, it can efficiently infect both dividing and non-dividing cells, and mediate transgene integrate into cell genome to achieve sustained and stable gene expression. Derived from the HIV-1, lentivirus (LV) is a member of retroviruses and has distinguishing efficiency in infecting primary cells and a major of cell lines. LV is a kind of positive-strand RNA virus, after infection, its genomic RNA reverse transcribed into DNA and then integrated into the host cell genome by reverse transcriptase and integrase. Protein was produced in the cytoplasm after the integrated DNA transcribed into mRNA. Lentiviral vector has many advantages over other vectors including high-efficiency infection of dividing and non-dividing cells, such as brain cells, liver cells, hematopoietic stem cells, bone marrow mesenchymal stem cells, macrophages; and long-term stable expression of transgene for the reason that the transgene irreversibly integrate in the genome and divisive with the division of the genome.At present, virus vector has becomea powerful tool for gene expression both in vivo and in vitro. As reported, lentiviral vector has been successfully used to produce decigram quantities of recombinant proteins with high purity and activity in human cell lines. To establish the technology of lentiviral vector-mediated gene expression in HEK293T cells to achieve long-term and effective production of recombinant protein, the most important thing is to find out a proper vector. Here we first evaluated the stability and expression level of transgene mediated by 5 different lentiviral vectors in HEK293T cells. Then the optimal lentiviral vector carrying HCV E1 gene is used to transduce 293T cells and recombinant cell lines are establish by limiting dilution. Five different vectors, pFIN-EFla-GFP-2A-mCherH-WPRE containing EFla promoter and HS4; p’HR.cppt.3’1.2kb-UCOE-SFFV- eGFP containing SFFV promoter and UCOE, pTYF-CMV (β-globin intron)-eGFP containing CMV promoter and P-globin intron, pTYF-CMV- eGFP only containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter, were constructed, packaged into lentivirus and titered, then 293T cells were infected in MOI=1000 (1000 viral genomes per cell). The transduced cells were passaged once every three to five days at a ratio of 1:10. Expression level and stability of GFP was evaluated using fluorescent microscopy and flow cytometry.Resμlts shows the titer of the 5 vectors quantified by real-time PCR were 2.8×108,3.7×108,2.5×108, 1.0×109,2.5×108vg/ml, respectively. We can see the GFP signal clearly at 3 days post-transduction in all five vectors and GFP expressions reached a peak between passages 3 to 5 and then lasted for more than 5 weeks in a stable level. Furthermore, the survival rates of all transduced cells were more than 80% at 5 or 9 weeks post-transduction. At five weeks, the mean positive rate of GFP was 22.7±3.3%,5.8±0.4%,54.1±3.7%,57.6±7.8% and 38.7±1.1%, respectively; while the mean fluorescent signal intensitie (MFI) was 6391.7±1030.4,1436.0±1.4, 21845.7±1959.0,26596.7±3900 and 9467.7±134.8, respectively. At nine weeks post-transduction, the mean positive rate of GFP was 27.6±6.9%,5.9±0.2%, 76.5±2.0%,91.7±1.7% and 38.2±3.9%, respectively, and the MFI was 1985.7±67.4, 386.5±5.0,12814.7±1703.6,21192±882.7 and 1664.7±113.4, respectively.The above results showed that the transduction efficiency of lentiviral vector containing the CMV promoter (with or without β-globin intron) is the highest in HEK-293T cells amongst the 5 different vectors, while those with the insulator and UCOE did not showed any enhancement in GFP expression level or stability. These findings provide valuable insights that the vector contain CMV promoter is an optimizing lentiviral vector for recombinant protein production in HEK 293T cellsIn order to further verify the expression level of complex heterologous protein mediated by lentivirus vector containing CMV promoter, we constructed a HCV E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was transduced to 293T cells, and the recombinant cell lines with stable expression level of E1 protein were established by limiting dilution. Gene expression in the transduced cells was evaluated by Western Blot using an anti-HCV polyclonal antibody.The coding sequence of HCV E1 was successfully cloned into the optimized pLV-CMV-eGFP vector, resulting in the pLV-CMV-E1 vector.293T cells were transduced with LV-CMV-E1 and clonal cell lines of HCV E1 were selected by limiting dilution. A representative recombinant cell line was generated, with stable and robust E1 expression for at least 9 weeks and no significant difference in morphology, compared with untreated 293T cells. |
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