生长抑素和皮质抑素双表达DNA疫苗免疫杂交水牛犊牛的促生长效果研究

试验旨在探究生长抑素（SS）和皮质抑素（CST）双表达DNA疫苗免疫杂交水牛犊牛的促生长效果及疫苗本身的安全性。将鉴定正确的pVGS/2SS-2A-S/CST-asd质粒电转入减毒猪霍乱沙门氏菌C500构建SS和CST双表达活载体疫苗，喷鼻免疫杂交水牛犊牛后观察其对犊牛生长性能的影响。选取体重相近的2~6月龄杂交水牛犊公牛24头，随机分为低（TL）、中（TM）、高（TH）剂量组和PBS对照组（NC），每组6头，初次免疫（简称初免）持续3 d，间隔2周后用相同剂量加免一次。结果显示，初免后2周便可产生抗SS和CST抗体，以TL组最佳，表现为更高的抗体水平和阳性率，7周时各试验组阳性率呈下降趋势；TL和TM组的平均日增重在初免后2和7周均高于对照组，但各组间差异均不显著（P>0.05），而抗体阳性组（P）的平均日增重在0~2周显著高于阴性组（N）（P<0.05）。相关激素的检测结果显示，在初免后2和7周，试验组生长激素（GH）和胰岛素生长因子1（IGF-1）的水平均高于对照组，且以TL组表现最佳，除初免后7周GH与对照组无显著差异外，其余均极显著高于对照组（P<0.01）；初免后2周抗体阳性组极显著高于阴性组（P<0.05）。细胞因子检测结果显示，在初免后2周，试验组白细胞介素4（IL-4）含量高于对照组，且TL和TM组均极显著高于对照组（P<0.01），尤以TL组最高，免疫后7周TL和TH组均显著高于对照组（P<0.05），而免疫后2周对照组免疫干扰因子γ（INF-γ）含量极显著高于试验组（P<0.01），免疫后7周对照组INF-γ含量显著低于TL和TH组（P<0.05）；阳性组IL-4水平在免疫后2周、INF-γ水平在免疫后7周均显著高于阴性组（P<0.05），其他各组间差异不显著（P>0.05）。对血清中血糖（GLU）、总蛋白（TP）、总胆固醇（CH）和尿素氮（BUN）水平进行测定发现，除免疫后2周总蛋白含量TL组显著高于对照组外（P<0.05），其他指标各组间差异均不显著（P>0.05）。对免疫后的水样、土样和粪样进行目的基因扩增发现，在PCR灵敏度范围内均未检测到，证实了疫苗的安全性。由上述结果可知，疫苗免疫后能取得良好的免疫效果，使机体产生显著的免疫应答，以低剂量组效果最佳，且没有对环境产生不良影响。

Study on the Growth Promoting Effect of Crossbred Buffalo Calves Immunized with DNA Vaccine of Co-expression of Somatostatin and Cortistatin

The purpose of this study was to investigate the growth promoting effect and the safety of the vaccine in crossbred buffalo calves immunized with somatostatin and corticostatin co-expression DNA vaccine.The identified plasmid of pVGS/2SS-2A-S/CST-asd was electroporated into attenuated Salmonella Choleraesuis C500 to construct the co-expression live vector vaccine of somatostatin and corticostatin,and evaluate the effect on the growth performance of the crossbreds buffalo after nasal immunization.Twenty four 2-6 months of age crossbred buffalo calves with similar body weight were randomly divided into 4 groups immunized with the vaccine in low dosage group (TL),middle dosage group (TM),high dosage group (TH) and negative control group (NC).Immunization was performed once a day for 3 consecutive days,and a booster was given 2 weeks late.The results showed that the antibody of SS and CST could be produced on 2 weeks after the primary immunization,and TL group was the best,showing higher antibody level and positive rate.At 7 weeks,the positive rate of experimental group showed a downward trend.The daily gain of each experimental group was higher than that of control group at 2 and 7 weeks after the primary immunization,but there was no significant difference between experimental groups (P>0.05),while the average daily gain of antibody positive group (P) was significantly higher than that of negative group (N) at 2 weeks (P<0.05).The results of related hormones showed that the levels of growth hormone (GH) and insulin growth factor 1 (IGF-1) were higher in experimental groups at 2 and 7 weeks after the primary immunization.In each experimental group,TL group showed the best performance,both extremely significantly higher than that of control group except GH level at 7 weeks after the primary immunization (P<0.01),antibody positive group was also significantly higher than negative group (P<0.05).Cytokine test results showed that the interleukin 4 (IL-4) level in experimental groups at 2 weeks after the primary immunization was higher than that of control group,and the IL-4 level in TL and TM groups was extremely significantly higher than that of control group (P<0.01),especially in TL group.The IL-4 level in TL and TH groups at 7 weeks after the primary immunization was significantly higher than that of control group (P<0.05).At 2 weeks after the primary immunization,the level of immune interferon γ (INF-γ) in control group was extremely significantly higher than that of experimental groups (P<0.01),but at 7 weeks after immunization was significantly lower than that of TL and TH groups (P<0.05).The levels of IL-4 and INF-γ in antibody positive group were significantly higher than that of negative group at 2 and 7 weeks after the primary immunization (P<0.05),respectively,and there was no significant difference among other groups (P>0.05).The levels of blood glucose (GLU),total protein (TP),total cholesterol (CH) and urea nitrogen (BUN) in serum showed that except that the level of total protein in TL group at 2 weeks after the primary immunization was significantly higher than that of control group (P<0.05),there was no significant difference of other indexes among each group (P>0.05).In order to confirm the safety of the vaccine,the genes of water,soil and fecal samples after immunization were amplified,which were not detected in PCR sensitivity range.The results indicated that the vaccine could achieve a good immune effect,make the body produce a significant immune response,and the effect of the low dosage group was the best.The vaccine did not cause adverse effects on the environment.