| The research was studied from two aspects of engineering bacteria preservation and immunity for the application of new somatostatin with attenuated Salmonella as carrier:The first part aimed to explore the methods of improving the effect of the DNA vaccine preservation through the comparison of several methods of preserving effect, and the second part was to discuss the effect and safety of cows of spray immunity with novel somatostatin DNA vaccine in late lactating from immune dose, in order to provide the experimental basis for the application of somatostatin gene vaccine in dairy cows.1. the preservation methods of engineered bacteriaThe initial concentration 8.43×1010cfu/mL of 600mL pVGS/2SS-asd liquid bacteria after fermentation and centrifugation in accordance with cryogenic liquid preservation, low-temperature drying preservation and freeze-dried preservation, divided into three equal parts, the survival rate of bacteria was measured with the viable plate count method. The cryogenic liquid storage effects were investigated under the different conditions of pH (6.8, 7.0,7.4,7.8), protective agent (sucrose, glucose, trehalose) and concentration(3%,5%,10%), the concentration of bacteria (8.43×1010cfu/mL,8.43×109cfu/mL,8.43×108cfu/mL); The low-temperature drying storage effects of adding different bacteria liquid volume (1mL,2mL, 4mL,8mL) to 100g bran were discussed; The freeze-dried preservation effects of different cryoprotective agents (A, B, C) and initial bacteria concentration (8.43×1010cfu/mL, 8.43×109cfu/mL,8.43×108cfu/mL) were investigated. The results showed that, the highest survival rate of cryogenic liquid preservation was pH7.0,3%sucrosed and bacteria concentration 8.43×1010cfu/mL in different factors; the bacteria survival rate of 1% initial concentration was higher than others, but the survival rate was only 0.31%, the method was not suitable for bacteria preservation; the bacteria survival rate was high with formulation A as cryoprotectant, and the higher the initial concentration the more conducive to the bacteria preservation in the freeze-dried preservation. Because the bacteria survival rate of cryogenic liquid preservation and freeze-dried preservation was 50.2% and about 15% respectively, the cryogenic liquid preservation is better than freeze-dried preservation in 15 days (pH7.0, without sugar, bacteria concentration 8.43×1010cfu/mL), and the freeze-dried preservation (cryoprotective agent A, the initial bacteria concentration 8.43 × 1010cfu/mL) was best 15 days later as the bacterial survival rate of cryogenic liquid preservation was significantly decreased.2. the study of cows immunized with somatostatin gene vaccine in late lactationIn order to investigate the effects of immunization against a novel somatostatin DNA vaccine—pVGS/2SS-asd on safety and lactation performance,80 cows in late lactation with accordant genetic background and milk production were divided into 4 groups,20 per group. The concentration of liquid bacterial was adjusted to 1×1010cfu/mL,2 times a day, cows were treated with three doses of high (15mL each time), in (10mL each time), low (5mL each time), and the control group (10mL each time)was treated with 10% aluminum hydroxide gel saline. The results showed that there was no security impact on cows immunized with the novel pVGS/2SS-asd somatostatin DNA vaccine after continuous observation for 7 days; certain antibodies still existed for 12 weeks after immunization, and medium dose group in the induction of the maximum antibody titer and the persistent high proportion of antibody positive cattle had obvious advantages than other dose groups. Each dose group could promote lactation, low-dose group was outstanding and increased by 2 kg/d compared to the control group at 12 weeks. |
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