Construction And Coexpression Bicistronic Expression Vector Based On FMDV 2A In Mammary Gland Cells

In this study, a mammary gland specific bicistronic expression vector pFIENβwhich containsβcasein 5'UTR as regulatory element and FMDV 2A was successfully constructed and its specific expression was detected in goat mammary epithelial cells. Firstly,βcasein 5'UTR was successfully cloned as regulatory element with IL-2 gene connected, and then the element was ligated with EGFP mediated by FDMV 2A thus pFIENβwas constructed. After Lipofectamine 2000 transfection into goat mammary epithelial cells with pFIENβ, RT-PCR and Western blotting analysis were conducted to detect the expression of the two genes. The research can lay foundations for coexpression of genes of interest such as hIL-2 and its synergistic pharmaceutical proteins through mammary gland bioreactor and the results of this research are as follows:1. Total RNA of human blood was successfully extracted and successfully subcloned through molecular cloning techniques such as RT-PCR, PCR, ligation, and transformation. It was demonstrated that IL-2 was successfully cloned after sequencing analysis of IL-2 thus could be applied for further research.2. Mammary specific coexpression vector regulated byβcasein 5'UTR was constructed to realize the expression of IL-2 and EGFP at the same time based on FMDV 2A.3,.Taking goat mammary tissue as material, primary cells of goat mammary epithelium were cultured by explant adherence method. Purified goat mammary epithelial cells were obtained by transferring the explants grown with mammary epithelial cells into another new plate and could be used for transfection in the further experiments as the cells grew well after cell passage.4. Liposome transfection was conducted and the optimum time for observation after transfection was determined. It is clear that transfection was successfully conducted for partial cells showed green fluorescence under fluorescence microscope after 2 days. Although RT-PCR certificated the the transcription of hIL-2 and EGFP, protein expression analysis was still conducted which demonstrated the expression of hIL-2, EGFP and the fusion protein. It is conclude that FMDV 2A mediates the cleavage of the fusion protein but not complete.