Development And Application Of The Retroviral Vector Based High-level Expression System

Retroviral expression system which consists of retroviral vector, envelop protein vector and packaging cell line is a potential and to be further improved expression system for recombinant protein. Transcriptional active genome regions are the preferred targets for retrovirus integration. Furthermore, VSV-G protein enables this system a broader host range and makes virus integration more efficient. It is more probable to obtain high-level expression cell line with infection of high titer virus.The main goal of this dissertation is to develop and evaluate the retroviral vector based expression system for recombinant proteins, and construct a recombinant HEK293 cell line expressing high-yield rhPC by using the developed expression system. Meanwhile, attempt to optimize the retroviral vector and enhance the efficiency of the expression system is made through introducing the regulating elements into the retroviral vector.A recombinant retroviral vector, PQCXIN/EGFP containing enhanced green fluorescence protein (EGFP) was constructed. The packaging cell line, GP2-293 was co-transfected with PQCXIN/EGFP and pVSV-G.. And recombinant PQCXIN/EGFP virus with infectious titer up to 1.61×107GTU/ml was obtained by ultra centrifugation. Both HEK293 cells and CHO-K1 cells were infected with recombinant PQCXIN/EGFP virus or transfected with pcDNA3.1/EGFP plasmid, respectively. The relative fluorescence tensity (RFT) from the EGFP positive cells was analysized by flow cytometry. The RFT from the recombinant PQCXIN/EGFP virus infected cells was about two times of the RFT from the cells transfected with pcDNA3.1/EGFP plasmid.The infectious titer and infection efficency are the two important factors for foreign gene expression in the retroviral vector based expression system. By using a two-step transfection protocle, GP2-293 cells were transfected with PQCXIN/EGFP and then, the GP2-293 cells exhibiting efficient EGFP expression were transfected with pVSV-G., resulted in about 50% increase in virus infectious titer. Furthermore, Polybrene at a concentration of 4 to 8 mg/ml enhanced the efficiency of infection to some extent.The effects of repeated virus infection on foreign gene expression in the retroviral vector based expression system were investigated as judging from the EGFP expression level. It was shown that the RFT from the cell population repeatly PQCXIN/EGFP virus infected up to four rounds was about 3.5 times higher than that of cell population merely infected with PQCXIN/EGFP virus one round. Moreover, after several passages, the RFT from the cell population repeatly PQCXIN/EGFP virus infected up to four rounds decreased 22%, whereas the RFT from the cell population transfected with pcDNA3.1/EGFP plasmid decreased 69%. These indicated that repeatly infected with PQCXIN/EGFP virus could be a method of choice for improving EGFP expression efficiency and stability.The efficiency of foreign gene expression in both PQCXIN/rhPC virus infected cell population and pcDNA3.1/EGFP plasmid transfected cell population was further judged by rhPC ELISA. The expression of rhPC in PQCXIN/rhPC virus infected cell population reached as much as 326.4ng/106cells/day, whereas the expression of rhPC in pcDNA3.1/EGFP plasmid transfected cell population was only 8.6ng/106cells/day.By replacing the neo gene with EGFP gene in the PQCXIN/rhPC vector and linking the EGFP gene to rhPC downstream via IRES, a PQCXIN/rhPC/EGFP vector, favouring high yield rhPC producing cells screening was constructed. A HEK293 cell line with rhPC expression capacity up to10.7μg/106cells/day was obtained through limited dilution clone and rhPC ELISA.To further enhance the foreign gene expression in the retroviral vector based expression system, the effect of regulating elements including promoters, enhancers, RNA stability elements, position effect elements on EGFP expression in pcDNA3.1/EGFP plasmid transfected cell population was examined. Among the reglating elements tested, RESE enhanced EGFP expression up to 1.58 fold. The effect of RESE on foreign gene expression in pcDNA3.1/EGFP plasmid transfected cell population was further proved by tPA, Pro-uk and rhPC expression, and a 5 times increase in rhPC expression was observed in pcDNA3.1/rhPC/RESE plasmid transfected cell population as comparing with the pcDNA3.1/rhPC plasmid transfected cell population. A HEK293 cell line with rhPC expression capacity up to16.9μg/106cells/day was obtained through limited dilution clone. A recombinant retroviral vector, PQCXIN/EGFP/RESE harbouring RESE has constructed at present, and the effect of RESE on EGFP expression in the retroviral vector based expression system is to be examined. In summary, these results demonstrated that the retroviral vector based expression system is a potential and to be further improved foreign gene expression system, and provided a foundation for rhPC research and development.