Construction And Evaluation In Immunogenicity And Safety Of Somatostatin DNA Vaccine Without Antibiotic Resistance Gene

A series of techniques were used in this study, such as gene cloning, cell culture, protein expression in vitro, Confocal microscopy, ELISA, RT-PCR and paraffin section. In order to construct somatostatin eukaryotic expression plasmid pVGS/2SS-asd without antibiotic resistance gene, a balanced lethal system was introduced into somatostatin DNA vaccine pGM-CSF/SS. Then, the plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain （with double deletion of crp and asd genes） by electroporation, and the recombinant strain C500 （pVGS/2SS-asd） was orally and intra-muscularly immunized against mice. The immunogenicity 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. Construction and identification of somatostatin eukaryotic expression plasmid without antibiotic resistance geneThe GS/2SS fusion gene amplified from pGM-CSF/SS plasmid was cloned into pVAX-asd vector without antibiotic resistance gene to obtain plasmid pVGS/2SS-asd. Subsequently, the M4GFP gene was then fused into 3’ end of GS/2SS gene in the proper reading frame to construct plasmid pGS/2SS-M4GFP-asd harboring enhanced green fluorescent protein （M4GFP） sequence. The insertion site, direction and sequence of the genes were identified to be correct by restriction endonuclease digestion and sequencing. The Confocal microscopy result showed that the green fluorescence was detected after 48 h of transfection and fusion protein GS/2SS-M4GFP was observed in the cytoplasm. RT-PCR result showed that the GS/2SS somatostatin gene could be detected. ELISA analysis indicated that all the fusion proteins possessed the immunologic competence of SS. This result indicated that the novel constructed plasmids could produce the fusion protein possessing the immunologic competence of SS in mammal cells.2. The stability analysis of recombinant strain C500 （pVGS/2SS-asd） in vitro and vivoThe plasmid pVGS/2SS-asd was transformed into Salmonella enterica sv. Choleraesuis C500 strain by electroporation, and formed SS DNA vaccine C500 （pVGS /2SS-asd） . Strains were passaged 50 generation in vitro, and the results showed plasmid pVGS/2SS-asd was stable in vitro culture. To investigate the bacterial colonization in organs and plasmid stability in vivo, mice were orally and intra-muscularly immunized with C500 （pVGS/2SS-asd）. Results showed the strains could be detected in lung, spleen, liver after 2d of immunization. There was no strain to detect at day of 14. The colony PCR showed the target fragment could be amplified from strains. These results showed plasmid pVGS/2SS-asd was stable in vitro and vivo.3. Immune response and affecting factors of SS DNA vaccineTo optimize the immunization schedule of SS DNA vaccine, three different dose (0.2×10¹⁰ CFU per mouse, 0.2×10⁹ CFU per mouse, 0.2×10⁸ CFU per mouse) and booster （0,1） and interval （4w, 6w） were performed in the current study. Results of ELISA showed that the anti-SS-antibodies were detected in the experimental groups, and the titers of antibodies presented a tendency of increase along with the extension of immunization. The high dose with no booster group was received the high antibodies compared with other groups. For the mean body weight of mice, the high dose with no booster group was also obtained the better body gain than the other groups.To investigate the types of immune response, the Balb/C female mice were orally and intra-muscularly immunized with C500 （pVGS/2SS-asd） , the dose of all groups was 0.2×10¹⁰ CFU per mouse. ELISA method was used to detect the titer of IgG, IgA and IgG subtype. As a result, the experiment groups could induce IgG, IgG1, IgG2a and IgA antibodies, and the intra-muscular group was better than oral group. For the detection of IgG subtype, the titer of IgG1 was higher than that of IgG2a both in oral and intra-muscular groups. These results indicating that immunization of C500 （pVAX-asd） by oral or intra-muscular administration can induce both Th1 type and Th2 type response, and Th2 type response was better than Th1 type.4. Expression and distribution pattern of pVGS/2SS-asd in mice various tissuesThe Balb/C female mice were orally and intra-muscularly immunized with C500 （pVGS/2SS-asd）, the dose of all groups was 0.2×10¹⁰ CFU per mouse. Mice were killed at 2d, 5d, 10d, 20d, 30d and 60d after immunization and every time 4 mice were sacrificed to collect tissues and organs including brain, heart, liver, spleen, lung, kidney, intestine, muscle and ovary. RT-PCR method was applied to detect the distribution of GS/2SS gene in transcription level. As a result, the target fragment of GS/2SS was detected in the tissues of heart, liver, spleen, lung, kidney and intestine after 2d of oral immunization, respectively. The total tissues were detected the GS/2SS gene with the exceptions of brain and ovary after 5d of oral immunization. At 10d after oral immunization, the GS/2SS gene was detected only in liver tissue. For intra-muscular immunization, the tissues of heart, liver, spleen, lung, kidney and muscular were observed objective fragment at 2d after injection, the result was the same as oral immunization at 5d after immunization. At 10d after immunization, the GS/2SS gene was detected in liver, muscular and spleen tissue. There was no band detected in tissues including oral route and intra-muscular route after 20d of immunization. These results indicated that the expression and distribution pattern of the fusion protein was transiently, and the fusion protein could degrade rapidly.5. Toxicity assessment of SS DNA vaccine in miceKunming mice were orally and intra-muscularly immunized with recombinant vaccine C500 （pVGS/2SS-asd） by the dose of 0.4×10¹⁰ CFU、0.4×10¹¹ CFU、0.4×10¹² CFU. Blood was collected at 4h、24h、1week、6week after immunization to detect hematology. Tissues such as heart, liver, spleen, lung, kidney, intestine, muscle, ovary or testis, brain were obtained to perform the paraffin sections. The hematology determination was detected with whole blood of mice and there was no significant difference among all groups. The results of paraffin sections showed the vaccine strain was no toxicity.At the termination of Balb/C mice （part 3）, the mice were killed to collect the tissues of heart, liver, spleen, lung, kidney, intestine, muscle, ovary, brain. 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.