Construction And Expression Of Recombinant Baculovirus Expression Vetor Coexpressing HA1 Gene From Subtype H5 Of Avian Influenza Virus And Mature Chicken Interleukin-18 Gene

The gene encoding interleukin-18 (IL-18) was firstly cloned by Okamura in 1995. IL-18 plays important roles in antitumor, anti-infection and immunoloregulation. And it has potential prospect in immunologic therapy and adjuvant. With the development of prologue industry in China, the poultry's disease especially Avian Influenza (AI) is becoming severity. So a newly vaccine and its adjuvant are urgently demand to research. And major antigen determinants of AIV lie in HA1 gene, so HA1 protein is the ideal electing antigen of studying gene engineering subunit vaccine. The gene encoding mature Chicken IL-18(mChIL-18) was cloned and expressed in our laboratory in order to prevent and therapy the poultry's diseases. In the present study, the efficiently expressed mChIL-18 protein with biological activity was obtained. It was included two parts as following:Experiment 1: Construction of Recombinant Baculovirus Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Mature Chicken Interleukin-18 GeneThe hemagglutinin(HA) gene of Avian Influenza Virus(AIV) subtype H5 and mature chicken interleukin-18(mChIL-18) gene were amplified by designing two pairs of primers according to the sequence published on GenBank.Recombinant baculovirus coexpressing HA1 gene from AIV subtype H5 plus chicken interleukin-18 gene were constructed by inserting cDNA copy of HA gene and cDNA copy of mChIL-18 gene into the baculovirus expression plasmid pFast Bac^TMDual,furthermore cDNAs respectively inserted into the downstreams of P10 promoter and PH promoter.Finally,the constructing eukaryotic expression plasmid pFastBac^TM dual-HA1-mChIL-18 was transposited into Bacterium Coli DH10Bac,which is based of next step insect cell's transfection.The construction of this recombinant plasmid is benefic to investigate the effect of mChIL-18's immunologic enhancement and study a new AIV recombinant vaccine. Experiment 2: Expression of Recombinant Vetor Coexpressing HA1 Gene from Subtype H5 of Avian Influenza Virus and Chicken Interleukin-18 GeneAfter constructing pFastBac^TM dual-IL-18-HA1 plasmid ,we make it transformate into DH10Bac competent cells.Then transformations are spreaded on fresh LB plates and the recombinant bacmid DNA is extracted.On the basis of M13 universal primers(The bacmid contains M13 Forward and M13 Reverse priming sites flanking the mini-attTn7 site within the lacZα-complementation region to facilitate PCR analysis),we analyze the recombinant bacmid DNA by PCR.Through transfecting insect cells, trcombinant baculovirus is harvested.Then we gain the high performance expressing product of IL-18 and HA1,which is benefic to investigate the effect of mChIL-18's immunologic enhancement and study a new AIV recombinant vaccine.