Secretory Expression And Functional Identifications Of A Porcine IFN-? In Pichia Pastoris

Porcine Interferon gamma interferon?pIFN-?? is the sole member of type ?interferon, mainly produced by activated T cells and NK cells, which having broad-spectrum antiviral activity and immunoregulation function. The construction of pIFN-? genetic engineering bacteria that have high-level expression, high activity and easy to purification, as well as the developing of pIFN-? related drugs, will have a great significance to reduce the use of antibiotic of pig-breeding industry, lack of effective antiviral antitumor agent, improve pork quality.In this study, m RNA sequence NM₂13948 posted on the NCBI was choosed as the pIFN-? coding sequence and Methylotrophic yeasts X33 was used as the expression host to express pIFN-?, the gene sequence of pIFN-? was also optimized. pIFN-? Pichia pastoris secreted expression vector p PICZ?A-pIFN-? ? p PICZ?A-pIFN-?-his tag and pIFN-?C-terminal mutant Pichia pastoris secreted expression vector p PICZ?A-pIFN-?? ?p PICZ?A-pIFN-??-his tag were constructed respectively. After that, SDS-PAGE and Western-blot were analyzed the expression of pIFN-? in different secreted expression vectors. However, the protein of pIFN-?-his tag that expressed in the first time can not be purified by nickel-chelate chromatography and can not be detected by Western-bolt; the protein of pIFN-? peptide chain C-terminal mutant pIFN-??-his tag that expressed in the second time can be purified by nickel-chelate chromatography and successfully detected by Western-bolt; The four kinds of proteins that expressed in twice were analyzed by SDS-PAGE, the gel showed the molecular size of four proteins: pIFN-??-his tag> pIFN-??>pIFN-?-his tag ? pIFN-?. It indicated that the first constructed pIFN-? secretion expression vector failed to give a complete expression of secreted C-terminal pIFN-?, while the second constructed pIFN-? secretion mutant expression vector was able to express complete C-terminal pIFN-?? successfully.After the purification of above four types protein, the purified proteins wereco-cultured with IPEC-J2?Porcine intestinal epithelial cell-J2?. In order to compare the stimulating activity differences of four proteins to the cell, the m RNA expression levels of downstream gene Mx1 and OAS1 regulated by IFN-?, and the genes PKR not regulated by IFN-? was analyzed. The results showed that four proteins could significantly improve the expression level of Mx1 and OAS1, however, they could not significantly improve the level of PKR; The transcription level of Mx1 and OAS1 in proteins expressed by the mutant expression vector increased more sharply than those without mutation expression vector,and the difference was significant. It suggested that the cell stimulating activity of pIFN-?expressed by mutant expression vector was stronger than that expressed by previous mutation expression vector. Through determination, the expression of pIFN-??-his tag in the highest-producing strain that constructed in the research was up to 536.4 mg/L, the level of expression of interest protein was satisfactory.In summary, the secretion expression of pIFN-? in Pichia pastoris, and the modification of pIFN-? C-terminal peptide chain can yield a complete protein C-terminal,and can be significantly improved its cell stimulating activity, it is very necessary. The study laid the foundation for the efficient and high-yield pIFN-? secretion expression in Pichia pastoris, and its application in the disease prevention and treatment in pig farming.