Improvement Of Recombinant Protein Expression In Baculovirus Vector System

Baculovirus expression vector system(BEVS)is one of the most valuble expression systems.As an important part of BEVS,there are two most widely used commercial insect cell lines,Sf9 and BTI-TN-5B1-4(High Five).Despite the suspension culture of them has developed relatively matural,there are some problems such as low cell viability,unstable cell proliferation,and vability decline,still exist during cell culture,which is detrimental to the production of recombinant baculovirus and expression of recombinant proteins.In this work,based on the insect cell suspension culture in shake flask,the improvements of cell culture process and recombinant baculovirus preparation were researched.Further,the protein expression processes of hTPO and Fiber-2 were established,which provided technical support for the recombinant protein production based on BEVS.First,the suspension culture processes of Sf9 and High Five in a 100 ml shake flask were invesgated.The optimal serum-free medium of both insect cells was IB-905 selected from several commercial culture media.In this medium,the optimal initial density,passaging period and culturing volume of two cells were determined.Besides those,High Five cells needed to be sealed with a 0.22 ?m air-permeable membrane to provide enough gas exchange.As for the routine maintenance of two insect cell lines,it was found that adding 1%fetal bovine serum(EBS)in IB-905 could effectively delay the cell declineSecond,the rencombinant baculovirus MultiBac-hTPO was constructed,and the proliferation and storage of baculavirus in different conditions were investigted.For virus proliferation,when infected time prolonged to 120 h,the titer of the baculovirus was higher in shaking culture.For virus storage,when stored at 4? for 3 months in the dark,the virus titer did not decrease significantly.When stored at-80? for a period,the virus titer was significantly reduced 50-80%and the degree of reduction was related to the serum concentration in the virus stockFinally,two foreign proteins,secreted protein(Human throid peroxidase,hTPO)and intracellular protein(flow adenovirus fiber protein 2,Fiber-2),were expressed in the BEVS.The result showed that compared to Sf9 cells,High Five cell had higher expression levels for two kinds of proteins.The optimal multiplicity of infection for hTPO expression was 0.1,and the post infection time was 96 h.As for Fiber-2,the optimal multiplicity of infection was 0.1,and the post infection time was 72 h.The optimal cell density at the time of infection was about 1.5×106 cells/ml.Two proteins were then producted based on the optimized BEVS.The hTPO,after expression,was purified by Ni-NTA affinity and the purity was more than 90%.After detecting by hTPO specific antibody,the purified hTPO showed well reactogenicity.The Fiber-2,after detected by Group I fowl adenovirus standard positive serum,also showed well reactogenicity,and the antigen titer reached 1:128.It showed that the recombinant Fiber-2 had a potential application in the development of group I fow adenovirus subunit vaccines.