Cloning Of Genetic Adjuvant GM-CSF And Immuno-Enhancement Of GM-CSF On Somatostatin DNA Vaccine

GM-CSF genes of pig and goat were cloned and fusion-expression plasmid (pGM-CSF/SS) harboring swine GM-CSF and somatostatin was constructed on the basis of pcS/2SS to study immuno-enhancement of GM-CSF on somatostatin DNA vaccine. A series of techniques were used, such as gene cloning, cell culture, protein expression in vitro, ELISA, RIA, semi-quantitative RT-PCR and regular chemical analysis. Experimental mice were immunized with pcS/2SS, pGM-CSF/SS and pGM-CSF+pcS/2SS at two routes of muscular injections (i.m.) and oral administration, respectively, and the immune response, GH and IGF-I secretion, GHR and IGF-I mRNA expression in muscle and liver tissues were detected. Subsequently, weaned piglets were orally immunized with pcS/2SS, pGM-CSF/SS and pGM-CSF+pcS/2SS delivered by attenuated Salmonella as carrier to study the effect of immuno-neutralization for growth-promotion, hormone secretion, SS antibody level and metabolite concentration. The current studies are tried to unlock the mechanism of immuno-enhancement of GM-CSF on SS DNA vaccine and the regulation of animal growth axis, to provide theoretic base for the clinical application of SS DNA vaccine.1 Cloning, sequencing and expression of GM-CSF genecDNAs encoding pig and goat GM-CSF were cloned and sequence analysis showed that they were 435 bp in length and encoded a predicted mature protein of 144 amino acids and the majority of the signal peptide. Compared with ovine, human, bovine, murine and canine GM-CSF, homology of the swine GM-CSF coding sequence was, respectively, 86.2%, 80.5%, 81.7%, 69.7% and 81.8% at the nucleotide level, and 78.5%, 71.3%,70.3%, 55.9% and 71.5% at the amino acid level. Compared with ovine, porcine, human, bovine, murine and canine GM-CSF, homology of the goat GM-CSF coding sequence was 98.8%, 88.7%, 89.3%, 91.3%, 74.0% and 89.9% at the nucleotide level, and 97.2%, 79.2%, 81.8%, 82.1%, 58.6% and 73.6% at the amino acid level. Swine GM-CSF gene was cloned into pcDNA3.1(-) to construct eukaryotic expression plasmid (pGM-CSF). Western blotting...