Prokaryotic Expression of Porcine Interferon -βand Its Inhibition Effect on Porcine Epidemic Diarrhea Virus
Abstract: The porcine IFN-βgene was amplified by polymerase chain reaction. The amplified fragment was cloned into pGEM-T-easy vector and then sequenced. The result indicated that the cloned gene was a complete porcine IFN-βgene with the codes for signal peptide ,which had the identities of 100% with the poIFN-βgene published in the GenBank. Another pair of primers was designed to sub-clone the gene coding porcine IFN-βmature protein and one rare codon near 5` terminus was modified to the biased codons of E.coli. After sequencing, the sub-cloned IFN-βgene was successfully inserted to expression vector pRLC and expressed in E.coli, the expressed protein was about 17.3% of the total cell protein. Recombinant porcine IFN-βexpressed as inclusion body, which was dissolved in 6 mol/L guanidine chloride and subsequently re-natured by diluting with refolding buffer containing GSH and GSSG. In order to obtain pure protein, the re-natured poIFN-βwas purified by Sepha- crylS-200 chromatography. As a result, the purified product was verified to be of high cytokine activity by inhibiting the cyto-pathogenic effect, which is about 5.6x105U/mg. In addition, the inhibition effect of recombinant poIFN-βon PEDV was determined using CPE50 method. The results indicated that high concentration of recombinant poIFN-βcould completely inhibit PEDV on PK-15 cell line.