Expression Of Porcine Alpha Interferon Gene In Prokaryotic System And Its Biological Activity Analysis

Interferon (IFN) , a kind of cytokines induced by a series of revulsants, exhibits various biological actions on both single cell and cells including antivirus, antiproliferation and bioimmunolization. From the first report on its discovery in 1957 by Isaacs and Lindenmann, plenty of researches have been carried out, and most of them are focoused on the study of its production in large scale for the application in treatment. The boom of biotechnique enhanced this process. Likewise, the administration of porcine alpha interferon will be an efficient way to solve the severe problem caused by virus infection in the porcine breeding. In this study, theα-interferon gene of pig (Landraces) was cloned and expressed in Escherichia coli and Bacillus subtilis, respectively, and then the biological properties of the recimbinant proteins were assayed. The main results obtained were as follows:1) Clone primers 5Pifn and 3Pifn were designed accroding to the nucleotide sequences of porcine alpha interferon gene (M28263). The interferon gene was amplified by PCR using the genome fragment of porcine liver cells as template. The DNA fragment obtained was cloned into pGEM(?)-T Easy Vector to generate the recombinant plasmid pEGM-PoIFN. DNA sequencing result showed that the full length of PoIFN gene was 681 bp, which contained an ORF of 570bp encoding 189 amino acids with a signal peptide of 23 amino acids.2) A pair of primers containing restriction sites of EcoRI and NotI were designed to subclone PoIFN into the multi clone site (MCS) of pET-30a vector to construct the recombinant expression plasmid pET-PoIFN. Then the plasmid pET-PoIFN was transformed into E.coli BL21 (DE3), and the recombinant strain was selected and named E.coli[pET-PoIFN]. SDS-PAGE and ELISA demonstrated that the recombinant porcine interferon gene was expressed in E.coli BL21 (DE₃) successfully.3) Primers 5Bifn and 3Bifn were designed by introducing restriction sites of SalI and XbaI, respectively. The fragment obtained by PCR was digested with SalI and XbaI, and then ligated with SalI /XbaI-linearized pEBS1 to form pBES1-PoIFN. The recombinant expression plasmid was transformed into Bacillus subtilis and the recombinant strain was selected and named B.subtilis [pBES1-PoIFN]. The SDS-PAGE of recombinant strain showed a specific band of 19 kDa.4) Inclusion body of recombinant porcine alpha interferon expressed in E.coli BL21 was dissolved in 8mol/L urea and renatured by diluting with refolding buffer that containing GSH and GSSG subsequently. The antiproliferative and antivirus activities of the renatured protein were measured by cell cubation. As a result, the renatured protein showed to be of latency activity by inhibiting the proliferation of cancer cell, but it had no antivirus activity on VSV. Neither the recombinant porcine alpha interferon expressed in Bacillus subtilis showed any antivirus capacity.