Preliminary Study On Stable Expression Of Canine FGF21 In CHOK-1 Cell Line And Optimization Of Its Expression Conditions

Objective: To find an optimal transfection method,transfect the eukaryotic plasmid pCMV3-His-dFGF21 into CHOK-1 cells,and screen for a stable expression of canine fibroblast growth factor FGF21 with hygromycin B.In this way,the canine fibroblast growth factor FGF21 could be stably expressed in Chinese hamster ovary cells(CHOK-1).And some expression conditions were optimized to provide reference for the engineering expression of the fibroblast growth factor FGF21 in the next step.This study laid the foundation for the development of FGF21 pet medication.Methods: The transfection results of eukaryotic plasmid pCMV3-His-dFGF21 were compared by three different transfection methods: lipofection,calcium phosphate precipitation and electroporation.Western blot was used to verify the expression of protein,and then the most suitable transfection method was selected according to result.The concentration gradient of hygromycin B(50,100,150,200,250,300 ?g/mL)was designed for transfected cells to obtain the optimal concentration which was used to screen the stable cell line.The transfection effect were confirmed by RT-PCR and Western blot,the growth curve was plotted to monitor cell growth,and cell viability was monitored using CCK8 to evaluate cell status.Orthogonal test was designed for 3 expression conditions of serum concentrations(8 %,10 %,15 %),cell inoculum density(30 %,40 %,50%)and expression durations(36 h,48 h,72 h)to research expression of canine fibroblast growth factor FGF21.The expressed product after cell disruption was detected by SDS-PAGE,and the protein content was analyzed using Gel Quant Express software.According to the results,the optimal culture conditions were selected to culture greatly the stable cell line.The sonicated protein was purified by Ni-Agarose affinity column,and the purified canine FGF21 was eluted in 40,100,300,500 mmol/L imidazole.Results: Three transfection methods were compared to determine the best method is lipofection.Cell viability was detected by MTT assay to obain the optimal concentration of hygromycin B was 200 ?g/mL.The cell line expressing the canine fibroblast growth factor protein FGF21 was successfully screened out,and the transfection effect was confirmed by RT-PCR and WB assay,which proved the successful construction of the stable cell line.optimal expression conditions were optimized according to the orthogonal test: cell seeding According to the orthogonal experiment,the optimal expression conditions were optimized: cell inoculation density 40 %,serum concentration 15 %,expression time 72 h,and the target protein was successfully eluted and purified under 100 mmol/L imidazole.Conclusion:Cationic liposome was selected to transfect cells.The canine fibroblast growth factor FGF21 cell line was stably expressed by the hygromycin B screening.The target protein could be expressed efficiently after optimizing the culture conditions of the cell line,and the protein was purified to provide an experimental basis for further exploration of its function.