Co-expression Of Recombinant Adenoviruses Carrying PIL18,pGM-CSF,pIFNγ And PCV2ORF2 Gene And Immunogenicity Enhancement Study

In this test, we have studied the immune enhancement of three kinds of cytokines on porcine interleukin-18 (porcine interleukin 18, pIL18), porcine granulocyte macrophage colony-stimulating factor (granulocytemacrophage colony-stimulating factor, GM-CSF), porcine interferon-γ(Interferon gamma, IFNγ). Recombinant adenovirus DNA were constructed based on the following methods:establishing chimeric fragment of the above genes and circovirus structural protein coding gene(PCV2ORF2) with recombinent PCR methods,respectively.And the sequence of the chimeric gene was then subcloned into a shuttle vector of pShuttle-CMV, electro-transformation with adenovirus genome (Adeasy-1) of Escherichia coli BJ5183-Ad-1.After purification, recombinant DNA were transfected into AinD-293 cells and expression of fusion protein of recombinant adenovirus were obtained.The test was divided into three parts to analyse the immuno characterization of the expressed fusion protein:Test 1:Constrcuting chimeric genes of PCV2ORF2 and the above three cytokines'mature peptide gene with a genetic connection(linker) by overlapping extension PCR method,respectively.The chimeric genes were then cloned into pMD18-T Simple vector.Enzyme digesion and nucleotide sequencing results showed that the obtained chimeric gene fragments of pIL18-ORF2,pGMCSF-ORF2, pIFNγ-ORF2 were 1338bp,1194bp,1259bp,respectively,which were accordance with relevant sequences in Genebank and the original reading frame.Test 2:The three chimeric genes were sequenced and subcloned into shuttle vector of pShuttle-CMV.The recombinant vectors were transformed into Escherichia coli BJ5183-Ad-1 by electroporation.Purifying the recombinant adenovirus DNA and transfected them into AD293 cell.PCR identification,RT-PCR indentification and immunofluorescence of protein expression results showed that the three recombinant vectors which expressed fusion proteins were constructed succesfuly.Test 3:Comparing the immuno characterization between PCV2ORF2 recombinent adenovirus and the above chimeric genes' co-expression proteins through mice experiments.The results indicated that rAd-LF2,rAd-GF2,rAd-NF2 and rAd-Cap could induce humoral immune response in mice and produce good immun protection,moreover,the immune effects of rAd-LF2,rAd-GF2 and rAd-NF2 were better than rAd-Cap,among which rAd-NF2 showed a significant effect of immuno enhancement.Our data proved that cytokines could enhance immuno effects to a certain degree.The studies show that: in the context of successfully constructing the chimeric gene vector, we have obtained the correct expressed recombinant adenovirus under the premise of using adenovirus expression vector. Also through the immune test it can be seen: the immune effect of immune adjuvant, such as pIL18, pGM-CSF, pIFNy, is significant. And compared to a single recombinant ORF2 adenovirus expression product, the mmunogenicity and reactogenicity of fusion protein, which is expressed by chimeric gene recombinant adenovirus, are better.