Preparation Of Dual-targeting Recombinant Interferon Gamma And Anti-fibrotic Activity

Activation of hepatic stellate cells（HSCs）during hepatic fibrosis rapidly amplifies and synthesizes a large amount of extracellular matrix protein（ECM）and tissue metalloproteinase inhibitors,causing excessive deposition of ECM proteins in the liver tissue,thereby causing fibrosis.All kinds of anti-fibrotic drugs are faced with the predicament of low efficacy and toxic side effects in clinical application.Enhancing the targeting of fibrotic liver tissue and activated HSCs by drugs is the key to solving these problems.Interferon gamma（IFNγ）can significantly inhibit the proliferation of fibroblasts and reduce the production of collagen,but its short half-life and severe systemic side effects limit its clinical application.In view of the abnormal deposition of type I collagen in patients with hepatic fibrosis disease and the superior affinity of human placental growth factor 2amino acid 123-144 peptide(PIGF-2_123-144)for type I collagen,this study considers to use the PIGF-2_123-144 fragment for modification of IFNγ,increase its targeting to fibrotic liver and retention at the lesion site.And the Bicyclic PDGFβR-recognizing peptide（BiPPB）that specifically recognizes PDGFβreceptor that is highly expressed on the surface of activated HSCs replaces the receptor recognition functional region of IFNγand confers its targeting on activated HSCs,thereby avoiding the non-specific activity of IFNγ.We first fused PIGF-2_123-144 and BiPPB-mimIFNγpeptide,and obtained the IFNγrecombinant protein,which has a high affinity with type I collagen and then targeted to fibrotic liver,and specifically targeted on activated hepatic stellate cells.In this study,we first modified the coding DNA sequence of human PIGF-2_123-144,and constructed a prokaryotic expression plasmid for the GST-PIGF-2_123-144 fusion protein.After being expressed in large quantities and purified by affinity,the fusion protein was highly affinity to I type collagen by ELISA test.Then the GST-PIGF-2_123-144-BiPPB fusion protein,in which PIGF-2_123-144 and BiPPB were linked by 3 glycines or 6 glycines,was expressed by prokaryotic cells,and the two fusion proteins after purification showed high affinity for I type collagen.Subsequently,GST-mim IFNγand GST-BiPPB-mim IFNγproteins were expressed and purified.Under the same molar number,GST-BiPPB-mimIFNγsignificantly inhibited the expression of MF phenotypic marker genes in activated HSC-T6 cells when compared with GST-mimIFNγ.Finally,a GST-PIGF-2_123-144-BiPPB-mim IFNγexpression plasmid was constructed.After prokaryotic expression and affinity purification,GST-PIGF-2_123-144-BiPPB-mimIFNγprotein significantly reduced type I collagen production in cells.The IFNγfusion protein engineered in this article has a strong specificity,a small molecular weight,and is easy to prepare,especially with dual targeting of fibrotic liver and activated hepatic stellate cells.