| Previous studies have investigated the use of a wide variety of cytokines (interleukin-1 [IL-1] to IL-8, IL-12, interferon, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor) as vaccine adjuvants, mainly concentrated on the vaccines of human beings and mouse expeiment. Interferon-gamma (IFN- r) is a cytokine that plays a versatile role in the regulation of immune systems and defensive responses of host to various pathogens. It is engaged in regulating the biological activities, such as interleukin gene expression, cell proliferation, B cell differentiation, acute phase response to injury and inflammation, T cell activation, and neutrophill and macrophage differentiation. It has also been described to promote the growth of some murine hybridoma and plasmocytoma, as well as human myeloma.Now little is known about the effect of porcine IFN- y geneon the immunity and the immune responses to vaccination. And there still lack the systematic explorations on the transgenetic expression in vivo and the effect of porcine IFN- y gene on the immune responses induced by routine protein vaccination. Our experiment is conducted to clone porcine IFN- y gene from lymphocytes of the special Chenghua pig and systematically study its expressing effect in vivo on the immune system and immune responses of mice and porcine to routine vaccines. It is expected to develop a novel immune potentiator to increase the immunity and enhance the immune responses of mice and porcine to vaccines to elevate the immunoprotective efficacy against different infectious pathogens.Porcine IFN- Y cDNA from lymphocyte was synthesized by RT-PCR from mRNA extracted from blood lymphocytes of Chenghua pig, which were stimulated by 10ug/ml Con A in vitro. The porcine IFN- Y cDNA from lymphocyte was then subcloned into pMD18-Tvector and named as pUPIFN- r. The sequence of cloned porcine IFN-r cDNA was determined with M13/pUC sequence primer using BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, USA) by ABI PRISM 377 DNA sequencer. The length of the IFN- Y gene was 498 bp, encoding a peptide of 166 amino acids whose molecular weight is 19.2 KDa and pI is 10.29. The signal sequence of peptides is from 63 to 136 bp and the mature peptides from 137 to 569 bp. By blasting the homologous sequences in GenBank databases, the sequence of porcine IFN-Y cDNA from lymphocyte is 98% homologus compared with the previously cloned porcine IFN- Y cDNA from spleen cell. The sequence of pig IFN- Y gene from lymphocyte was submitted to GenBank and registered as AF493993.The pUPIFN- Y was digested with BamH I, and then the insert was recovered from agarose gel using Agarose Gel DNA Extraction Kit. VR1020 was used to construct the eukaryotic expression plasmid, VR1020 was digested with BamH I, and the insert was ligated with the digested VR1020. The recombinant plasmid was named VPIFN- Y . VPIFN- Y was transformed into E.coli DHScc competent cells. The recombinant plasmid were extracted and digested with Kpn I to identifie the correct insert orientation. The ability of VPIFN- Y to express full-length porcine IFN- Y in vitro was confirmed by RT-PCR in COS7 cells that were transiently transfected with VPIFN- Y in the entrapment of Lipofectamine and by the biological activities of the expression products to increase remarkably the proliferation of lymphoblasts from the blood of porcine and the spleen of mice stimulated by Con A.270 BAL b/c female mice at the age of 6 weeks were divided into 18 groups and were vaccinated intramuscularly in the left and right quardriceps respectively with 30 pmol of naked VPIFN- Y and pUC18-CpG DNA, 6 pmol VPIFN- Y and pUC18-CpG DNA entrapped with cationic liposome and poly (lactide-co-glycolide) (PLG) nanometer particles, which were prepared in our lab. Half of them wereinoculated along with the trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas, and pig paratyphoid vaccine. Mice were bled on 7, 14,21 days after immunization to determine the dynamic changes of immune cel...
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