Construction Of Genetic Engineering Strain Expressing Fusion Protein IFNβ－HSA

The interferon (IFN) is one of the potent multifunctional cytokines family, which possesses a wide range of anti-viral, immunoregulatory and growth inhibitory properties. As a member of interferon family, interferonβhad been applied to treat many diseases, such as MS and hepatitis. To prolong the half-life of IFNβin vivo, we developed a long-acting IFNβ(IFNβ-HSA) by albumin fusion technology. The fused gene IFNβ-HSA was expressed in pichia pastoris. Confirmed it by Western blot and antiviral analysis, the fusion protein was purified preliminarily.PCR technology was employed to amplify the IFNβgene, with a pair of primers designed according to the nucleotides sequence of IFNβgene pressed in GenBdank and the template DNA extracted from human leukocyte. The IFNβgene recovered from PCR was cloned into plasmid pBluescriptâ…¡KS(+) at Salâ… and Hindâ…¢restriction enzyme sites, and then sequenced. The result demonstrated that the nucleotides sequence of cloned IFNβgene was in correspondence with IFNβcDNA pressed in GenBdank with accession number NM₀02176.After splicing IFNβand HSA genes in vitro by overlapping PCR technology, the fused gene was cloned into plasmid pBluescriptâ…¡KS(+) and then sequenced. The recombinant plasmid was named pBlu2KSP-IFNβ-HSA. The confirmed fused gene was recovered from the plasmid with restriction endounclease EcoRâ… and Notâ… , and then inserted it into expression vector pPIC9k to construct recombinant expression vector pPIC9k-IFNβ-HSA. The results showed that the cloned fused gene was in correspondence with our anticipation, and expression vector was constructed correctly.The expression vector pPIC9k-IFNβ-HSA linearized Salâ… restriction endonuclease was transformed into KM71 strain of pichia pastoris by electroporation. And the recombinant strains were screened by His limitation,G418 resistance and expression level by step to step. The genetic engineering strain with relative high expression level was identified by western bolt and antiviral analysis. After optimizing the time of inducement and the concentration of inducer, the secreted fusion protein IFNβ-HSA was purified by the isolated procedure of ultrafiltration and gel filtration chromatography, respectively. Finally, 12 strains were screened out from contained 3 mg/mL G418 MD plate, in which 4 strains could secrete fusion protein IFNβ-HSA in relative high level. Western blot analysis with HSA antibody showed that the fusion protein IFNβ-HSA was on 90kDa molecular weight. The specific activity of culture supernatant was about 640 IU/mL assayed by the standard antiviral activity test on WISH cells challenged with VSV virus. The fusion protein was purified to a purity of about 90%.