Cloning, Prokaryotic Expression Of Interferon-β Gene And Construction Of Its Eukaryotic Expression Vector In Pichia Pastoris And Further Identification Of IBV Receptor

Interferon(IFN) is a kind of cytokine with antiviral,anti-tumor and regulatory immunity functions.Since Isaacs and Lindenmann reported in 1957,the studies on IFN and its development and application never stopped.Now it has been utilized in the control of variable diseases for human beings.In 1980,IFN was classified into three categories,IFN-α,IFN-βand IFN-γ,by world health organization based on the antigenic specificity.Afterwards it was classified into two types on their different acceptors,typeⅠIFN and typeⅡIFN.TypeⅠIFN includes IFN-α,β,ω,τ,δetc,and typeⅡIFN only contains IFN-γ.Research about IFN-γis very hot during last decades and several products about recombinant IFN-γhave been applied in market.However,research on IFN-α,and IFN-βis not as much as that of IFN-γ.The IFN-βproducts for different specieses are very rare. Latest research showes that IFN-βpossesses multiple functions as one of the important cytokines.Although anti-virus mechanism is similar to IFN-α,IFN-βhas some special characters which are highly related to its anti-virus effect.The studies on the animal IFN began comparatively later in veterinary medicine.For cattle,various infectious diseases such as mastitis,foot-and-mouth disease, bovine tuberculosis,etc,caused by viral or bacterial pathogens not only adversely affect the development of national and regional cattle industries,but also endanger human health in the whole world.The research on IFN-βwill improve the disease control of cows by enhancing cow's immunity and establishing a defense mechanism against viruses and bacteria.Avian Infectious Bronchitis(IB) is a highly contagious viral infection of the domestic fowl caused by infectious bronchitis virus(IBV).It can lead to respiratory disease,kidney inflammation with low production and poor quality of eggs.The previous study in this lab has lauched some initial research on identification of the IBV natural receptor,HeLa and PK-15 cell monolayers that do not permit natural infection by IBV were transfected with gAPN-neo plasmid.The transfected cells became to be permissive to IBV.However,receptor function of gAPN in the natural host chicken was not studied. Thus,part of this dissertation continued to determine the expression of gAPN in chicken tissue and the interaction between IBV and the tissues.The main results were summarized as follows:1.Cloning and sequence analysis of bovine interferon-beta gene and its expression in E.coliThe total RNA was extracted from peripheral blood mononuclear cells(PBMC) which was isolated from bovine and induced with phytohemagglutinin(PHA),then the mature peptide of bovine interferon beta(mBoIFN-β,498bp) was amplified by RT-PCR. The result of sequencing demonstrated that the cloned mBoIFN-βgene had 100% homology at nucleotide level to that published on Genbank,Furthermore,the mBoIFN-βhad 59%,50%,51%,46%,49%～51%,36%～43%homology at amino acid level respectively to that of Sus scrofa,horse,feline,canis,human,and mice.The mBoIFN-βgene was inserted into pET-28a(+) and the recombinant plasmid was transformed into BL21 E.coli strain,and the expression was induced by IPTG.SDS-PAGE demonstrated that the protein with the size of 28.6 kDa) mainly existed in insolvable inclusion body. The best inducing time was 4 h after the addition of IPTG..2.Construction of eukaryotic expression vector of IFN-βin pichia pastorisTo highly express secreted bovine interferon-beta,the signal peptide was excised from the BoIFN-βgenes and the mature peptide(mBoIFN-β,495 bp) was cloned into the yeast-Escherichia shuttle vector pPIC 9K to construct secreting recombinant expression plasmid of pPIC 9K- mBoIFN-β.The pPIC 9K-mBoIFN-βwas linearized by SalⅠand co-transformed with ssDNA into Pichia pastoris cells GS115(defective with histidine) with LiCl.The transformants were selected with MD culture plates and the mBoIFN-βgene insertion was identified by PCR.The multicopy recombinant Pichia pastoris strain was selected by G418 resistance.3.The expression of gAPN in chicken,the IBV natural host In order to demonstrate the receptor function of gAPN in IBV natural host,this study used the semi-quantity RT-PCR to analyze the levels of gAPN gene transcription in chicken tissues.Meanwhile,the immuno-histochemistry was performed to confirm the expression of gAPN in chicken tissues.As a result,the tissues with gAPN gene transcription in descending order was as follows:ovary,oviduct,trachea,spleen,ileum,lung,liver,duodenum, kidney,jejunum,and heart.This distribution is in agreement with the tissue tropism of IBV.the immuno-histochemistry demonstrated that gAPN was expressed in ileum,? kidney,liver,heart,trachea.Besides,flow cytometry showed that chicken embryo cells expressed gAPN and was able to attach IBV.