Abstract:

The aim of the study was to generate a transgenic swine fibroblast cell line of pepsin- and trypsin-toleranceing. After culturing, transgenic yeast fermentation broth was collected to detect the enzyme activity under the condition of pH2.0 for pepsin treatments with different concentrations and pH7.0 for trypsin treatments with different concentrations, respectively. The expression vector containing phytase gene was packaged in lentivirus and then infected swine fibroblasts. After subculturing, positive cells were screened with flow cytometric approach and cultured. The results showed that more than 70% of phytase activity remained after treatment with pepsin and more than 80% activity was remained when treated with trypsin. Lentivirus was packaged with 3.75×10⁷ titer and transfected into porcine fibroblast cells, with 1.2% positive rate obtained. PCR assessment indicated that the exogenous gene was inserted into the chromosome of the swine fibroblast cells successfully. Moreover, RT-PCR evaluation suggested that the phytase gene expressed in the transcript level. In short, the study provides a fast and effective approach for preparing phytase transgenic cell line, as well as a foundation for further breeding transgenic pig.