Establishment Of A Antiprotease Phytase Transgenic Swine Fibroblast Cell Line

The application of phytase can effectively solve the problem of high phosphorus pollution in monogastric animals fecal. However, it is easier inactivation in feed processing. With the development of transgenic technology, it will be an effective way to solve this problem though producting phytase by monogastric animals endogenous bioreactor. In the generated transgenic phytase pig gene, the phytase gene appA is acidic and derived from Escherichia coli. So it is easier inactivation and plays a relative short role under the intestinal gastrointestinal alkaline conditions. The aim of the study is to generate a pepsin-and trypsin-tolerance transgenic phytase swine fibroblast cell line and lay a foundation for further breeding transgenic pig. This main results investigated are as follows:1. The phytase tolerence of pepsin and trypsin was detected. A pichia pastoris expression vector, pGAPZaA-Cafp, was established and transformed into the pichia pastorios cells X-33. Positive cells were obtained by increasing the concentration of zeocin in medium gradually. After culturing for72h, fermentation broth was collected to detect the enzyme activity in the condition of pH2.0under pepsin treatment and pH7.0under trypsin treatment, respectively. Results showed that more than70%of phytase remained active after the treatment with pepsin at pH2.0and over80%activity remained when treated with trypsin at pH7.0.2. Lentivirus was packaged. The expression vector was constructed and packaged in lentivirus and then infected293T cells. Cells supernatant were collected after infecting24h,48h and72h. Virus particles were obtained after purifying the cells supernatant. The titer was3.75×107tu/mL with dilution detection assay.3. Transgenic phytase pig fibroblast cell line was established. Lentivirus with high titer were transfected into porcine fibroblast cells and the infection efficiency was observed with the fluorescence microscope after72h. Flow cytometry sorting technology was used after passing three generations and positive transgenic rate was1.2%. PCR assessment indicated the exogenous gene was inserted into the chromosome of the swine fibroblast cells successfully. Moreover, RT-PCR evaluation suggested that the phytase gene expressed on RNA transcript lever.