Effect Of Somatostatin DNA Vaccine With Fusion-Expression Plasmid Harboring GM-CSF And IL-6 Gene On The Growth Of Mice

IL-6 gene was commonly used as immunopotentiators. In ord to discusse its immunologic enhancement at genetic immunization, a series of techniques were used in this study, such as gene cloning, cell culture, ELISA, RT-PCR. IL-6 gene of cattle was cloned in order to construct somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance gene, on the basis of somatostatin DNA vaccine pGS/2SS-asd. Then, the plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain (with deletion of asd genes) by electroporation, and the recombinant strain C500(pGS/2SS-IL6-asd) was intra-muscularly immunized against mice. The immunogenicity of fusion-expression plasmid (pGS/2SS-IL6-asd) harboring GM-CSF and IL-6 gene, and safety assessment were discussed here to develop gene immunization techniques to promote growth of animals and to accelerate clinical application of SS gene vaccines.1. Cloning and sequencing of IL-6 geneIL-6 gene of cattle was cloned by RT-PCR. The RNA of cells were stimulated and induced by ConA and PHA. cDNA encoding cattle IL-6 was cloned and sequence analysis showed that it was 624 by in length. Compared with the sequence of IL-6 gene of cattle publish in Genbank (X57317.1).Homology of the cattle IL-6 cloned coding sequence was 99.84%.Cattle IL-6 gene was cloned into pEASY-T1-simple to store.2. Construction and identification of somatostatin eukaryotic expression plasmid pGS/2SS-IL6-asd without antibiotic resistance geneThe IL-6 gene was then fused into 3'end of GS/2SS gene in the proper reading frame to construct fusion-expression plasmid pGS/2SS-IL6-asd harboring GM-CSF and IL-6 gene without antibiotic resistance gene. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. RT-PCR result showed that the GS/2SS-IL6 somatostatin gene could be detected.3. The somatostatin DNA vaccine pGS/2SS-IL6-asd without antibiotic resistance gene influence the growth of miceThe plasmid pGS/2SS-IL6-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500(pGS/2SS-IL6-asd). A total of 50 female Kungming mice were divided into 6 groups.Group A, B and C were immunized with pGS/2SS-IL6-asd by three different dose(1×1010CFU per mice,1×109CFU per mice,1×108 CFU per mice). Group D was immunized with pGS/2SS-asd(1×109CFU per mice). Group E was immunized with C500(1×109CFU per mice). Group F was immunized with PBS. Enhance immunization after two weeks from the first immunization. Blood collection was at 0,14,28,42 and 56 days. Results of ELISA showed that the anti-SS-antibodies were detected in the A to D groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high and middle dose group immunized with pGS/2SS-IL6-asd were received the high antibodies compared with the low dose group and pGS/2SS-asd group (p<0.01).Every week weight the body weight of mice. For the mean body weight of mice, the high dose group immunized with pGS/2SS-IL6-asd was also obtained the better body gain than the other groups. In conclusion, fusion-expression of IL-6 and GM-CSF can stimulate immune responses and enhance spleen lymphocyte proliferation responses of immunized mice to specific antigen. Immune response of fusion-expression plasmid of pGS/2SS-IL6-asd is superior to pGS/2SS-asd.4. Chromosomal integration analysisAt the termination of Kungming mice, the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, muscle and ovary. The genomic DNA was extracted, and then the genomic DNA samples were analyzed by sensitive PCR method. There was no evidence of integration to sensitivity of about 10 copy/μg DNA. If integration occurred at all, the frequency would be at least two orders of magnitude below the spontaneous mutation rate.