Construction of Foot-and-mouth disease virus 2A-based bicistronic expression vector and coexpression of two genes in goat mammary epithelial cells.

摘要

Using animal mammary glands as bioreactors for producing commercially important proteins is a cutting-edge direction in the field of biotechnology development and application. Dairy goats are an important dairy livestock, with roughage-resistance, fast propagation, long lactation periods and high milk production per bodyweight; these characteristics make dairy goats ideal for use as mammary gland bioreactors. Foot-and-mouth disease virus 2A (FMDV 2A) is an efficient viral cleavage element that mediates proteolytic cleavage independent of the presence of other FMDV sequences. It is often incorporated into recombinant vectors to generate cleavage in the presence of heterologous sequences. To achieve specific co-expression of two heterologous genes in goat mammary gland epithelial (GMGE) cells, a mammary gland-specific bicistronic expression vector, pFIE beta , containing the beta -casein 5' flanking sequence and FMDV 2A, was successfully constructed and the specific expression of human interleukin 2 (hIL-2) and enhanced green fluorescent protein (EGFP) was conducted in primary GMGE cells. Another bicistronic expression vector, pFIEC, driven by the cytomegalovirus promoter, was constructed as a positive control. In cells transfected with pFIE beta and pFIEC, RT-PCR verified the existence of recombinant fusion mRNA of hIL-2 upstream of EGFP within the FMDV 2A cassette fragment and western blot analysis showed the existence of the fusion between hIL-2 and EGFP. It is concluded that FMDV 2A generated specific co-expression of multiple genes for the first time in primary GMGE cells driven by the beta -casein promoter.