生长抑素和皮质抑素双表达DNA疫苗免疫杂交水牛育成牛的促生长效果研究

doi:10.16431/j.cnki.1671-7236.2021.04.023

摘要/Abstract

摘要： 试验旨在探究生长抑素（SS）和皮质抑素（CST）双表达DNA疫苗免疫杂交水牛育成牛的促生长效果，并对其安全性进行验证。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构建生长抑素和皮质抑素双表达活载体疫苗。将16头6~12月龄杂交水牛育成牛随机分为低（TL）、中（TM）、高（TH）剂量组及PBS对照组（NC），每组4头，试验组和对照组分别用双表达疫苗和PBS进行鼻腔免疫，初免时每天09：00和15：00免疫2次，连续免疫3 d，2周后以相同程序加强免疫1次，对免疫后不同时期的增重效果和免疫应答水平等进行监测。免疫应答结果显示，各剂量组免疫后均引起了免疫应答，TL组在初免后2周产生的SS抗体水平高于TM和TH组，而TM组SS抗体阳性率最高，为100%，优于TL （75%）和TH组（50%）；初免后2周产生的CST抗体水平则呈现完全相反的趋势，抗体水平随免疫剂量下降呈下降的趋势；初免后7周SS抗体和CST抗体水平和阳性率整体呈下降趋势，但也有部分牛出现抗体升高趋势；免疫因子检测结果显示，初免后2周TL和TM组IL-4水平极显著高于对照组（P<0.01），而免疫后7周对照组的IL-4水平显著高于TL组（P<0.05），与其他各组之间差异均不显著（P>0.05）；对照组INF-γ水平在初免后2周显著高于TL和TM组（P<0.05），免疫后7周对照组显著高于TL组（P<0.05），与其他各组之间差异均不显著（P>0.05）；对比抗体阳性组和阴性组发现，初免后2周抗体阳性组的IL-4水平极显著高于阴性组（P<0.01），初免后7周则显著低于对照组（P<0.05），而抗体阳性组INF-γ在初免后2和7周均显著低于阴性组（P<0.05）。增重效果检测显示，初免后2周各试验组平均日增重均显著高于对照组（P<0.05），而在其他各时期各组之间差异均不显著（P>0.05），抗体阳性组仅在初免后2周显著高于阴性组（P<0.05）。激素检测结果显示，各试验组生长激素（GH）和胰岛素样生长因子-1（IGF-1）在初免后2周均极显著高于对照组（P<0.01），而初免后7周TL组GH和IGF-1水平显著低于对照组（P<0.05），其他各组之间均差异不显著（P>0.05）；抗体阳性组的GH和IGF-1水平在初免后2周极显著高于阴性组（P<0.01），而初免后7周则极显著低于阴性组（P<0.01）。安全性检测结果显示，各时期采集的水样、土样、粪样中均未检测到目的片段，证实了疫苗的安全性。本试验结果表明，双表达疫苗免疫后机体能产生良好的免疫应答，能提高育成牛的平均日增重，且对环境未产生安全性问题。

关键词: 生长抑素; 皮质抑素; DNA疫苗; 杂交水牛; 育成牛

Abstract: The purpose of this study was to investigate the growth promoting effect and the safety of the vaccine in crossbred buffalo growing cattle 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.Sixteen 6-12 months old crossbred buffalo growing cattle were randomly divided into low dosages group (TL),middle dosages group (TM),high dosages group(TH) and negative control group(NC).At the initial immunization,immunization was performed at 09:00 and 15:00 twice a day for 3 d,and the same procedure was used to strengthen immunization once after 2 weeks.The experimental group and control group was immunized with co-expression vaccine and PBS respectively,and the effect of daily weight gain and immune response level in different periods after the primary immunization.The immune response results showed that all dose groups induced immune response after the primary immunization,the antibody of somatostatin (SS) in TL group was higher than that in TM and TH groups at 2 weeks after primary immunization,and the positive rate of SS antibody in TM group was the highest,which was 100% and better than that in TL (75%) and TH groups (50%).However,the antibody of cortistatin(CST) after 2 weeks of primary immunization showed a completely opposite trend,high dosages group showed the best performance,and with the decrease of immune dose,the antibody of CST also showed a downward trend.After 7 weeks of primary immunization,antibody of SS and CST and positive rate showed a downward trend,but some buffaloes showed an upward trend.In the aspect of cytokines,it was found that the interleukin 4 (IL-4) level at 2 weeks after the primary immunizationin TL and TM groups was significantly higher than that in control group (P<0.01).At 7 weeks after primary immunization,the IL-4 level in control group was significantly higher than that in TL group (P<0.05),and there was no significant difference with other groups (P>0.05).The level of immune interferon γ (INF-γ) at 2 weeks after the primary immunization in control group was significantly higher than that in TL and TM groups (P<0.05).The INF-γ level at 7 weeks after the primary immunizationin in control group was significantly higher than that in TL group (P<0.05),but there was no significant difference with other groups (P>0.05).The IL-4 level at 2 weeks after the primary immunizationin in antibody positive group was significantly higher than that in negative group (P<0.01),and was significantly lower than that in control group at 7 weeks after the primaryimmunization (P<0.05),but the INF-γ level at 2 and 7 weeks after the primary immunizationinin in the antibody positive group was significantly lower than that in the negative group (P<0.05).The weight gain effect test showed that the average daily gain of each experimental group was significantly higher than that of control group at 2 weeks after the primary immunization (P<0.05),but there was no significant difference among the groups in other periods (P>0.05).The antibody positive group was also significantly higher than the negative group at 2 weeks after the primary immunization (P<0.05).The hormone test results showed that growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels in the experimental groups were significantly higher than those in control group at 2 weeks after the primary immunization (P<0.01),while the levels of GH and IGF-1 in the low dosages group were significantly lower than those in control group (P<0.05),but there was no significant difference between the other groups (P>0.05),the levels of GH and IGF-1 in the antibody positive group were significantly higher than those in the negative group at 2 weeks after the primary immunization (P<0.01),and were significantly lower than those in control group at 7 weeks after the primary immunization (P<0.01).In the aspect of safety,no target fragment was detected in water,soil and fecal samples collected in each period.The results showed that the co-expression vaccine could induce immune response and promote the average daily gain of growing cattle,and no safety problem was detected.

Key words: somatostatin; cortistatin; DNA vaccine; crossbred buffalo; growing cattle

中图分类号:

S823.9

杨帅, 张欣欣, 周迪, 袁婧, 杨利国. 生长抑素和皮质抑素双表达DNA疫苗免疫杂交水牛育成牛的促生长效果研究[J]. 中国畜牧兽医, 2021, 48(4): 1361-1369.

YANG Shuai, ZHANG Xinxin, ZHOU Di, YUAN Jing, YANG Liguo. Study on the Growth Promoting Effect of Crossbred Buffalo Growing Cattle Immunized with DNA Vaccine of Co-expression of Somatostatin and Cortistatin[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1361-1369.

参考文献

[1] 朱兵山,张晓雪,魏趁,等.新疆天山某安格斯牛场安格斯牛生长发育规律分析[J].中国畜牧兽医,2018,45(6):1535-1542. ZHU B S,ZHANG X X,WEI C,et al.Analysis on growth regulation of Angus cattle in a Angus ranch of Tianshan in Xinjiang[J].China Animal Husbandry & Veterinary Medicine,2018,45(6):1535-1542.(in Chinese)
[2] 刘丽元,臧长江,周靖航,等.新疆昌吉地区荷斯坦奶牛生长发育规律分析[J].中国畜牧兽医,2015,42(8):2036-2041. LIU L Y,ZANG C J,ZHOU J H,et al.Analysis on growth and development pattern of Holstein cows in Changji area of Xinjiang[J].China Animal Husbandry & Veterinary Medicine,2015,42(8):2036-2041.(in Chinese)
[3] PASIERBSKI Z,WAWRZYNCZAK S,JOCZ B.Fattening characteristics and slaughter value of Black Pied cattle and their crosses having various proportions of Holstein-Friesian breeding,under various levels of feeding[J].Roczniki Naukowe Zootechniki,Monografie Rozprawy,1991,29:57-72.
[4] CANTALAPIEDRA-HIJAR G,ORTIGUES-MARRY I,SEOCHAT B,et al.Methionine-balanced diets improve cattle performance in fattening young bulls fed high-forage diets through changes in nitrogen metabolism[J].British Journal of Nutrition,2020,124(3):273-285.
[5] JU YR,BAEK YC,JANG SS,et al.Nutritional value and in situ degradability of oak wood roughage and its feeding effects on growth performance and behavior of Hanwoo steers during the early fattening period[J].Asian-Australasian Journal of Animal Sciences,2020,33(6):930-960.
[6] HAN Y G,HAN Y Q.WU J Y,et al.Immunisation of somatostatin DNA vaccine in ewes can affect serum somatostatin and prolactin levels in offspring lambs through maternal related hormones[J].Pakistan Journal of Zoology,2020,52(2):801-804.
[7] DONG F X,LUO X,YUAN D L,et al.Construction and evaluation of novel plasmid dna encoding somatostatin fused to a tissue plasminogen activator (tPA) in mice[J].Pakistan Journal of Zoology,2018,50(5):1875-1883.
[8] 罗璇.生长抑素和皮质抑素双表达DNA疫苗的构建和鉴定及作用机制研究[D].武汉:华中农业大学,2014. LUO X.Construction,evaluation and mechanism of a novel DNA vaccine dually expressing somatostatin and cortistatin[D].Wuhan:Huazhong Agricultural University,2014.(in Chinese)
[9] LUQUE R M,KINEMAN R D.Gender-dependent role of endogenous somatostatin in regulating growth hormone-axis function in mice[J].Endocrinology,2007,148:5998-6006.
[10] LUQUE R M,PEINADO J R,GRACIA-NAVARRO F,et al.Cortistatin mimics somatostatin by inducing a dual,dose-dependent stimulatory and inhibitory effect on growth hormone secretion in somatotropes[J].Journal of Molecular Endocrinology,2006,36:547-556.
[11] SPENCER G S G,GARSSEN G J,HART I C.A novel approach to growth promotion using auto-immunisation against somatostatin.Ⅰ.Effects on growth and hormone levels in lambs[J].Livestock Production Science,1983,10(1):25-37.
[12] 黄夺先,赵伟,孙益兴,等.生长抑素(Somatostatin)主动免疫山羊的增重效果[J].江苏农业学报,1991,4:37-40. HUANG D X,ZHAO W,SUN Y X,et al.Effect of somatostatin active immunization on weight gain of goats[J].Jiangsu Journal Agricultural Sciences,1991,4:37-40.(in Chinese)
[13] HAN L,LIANG A X,ZHANG J,et al.Evaluation of the VP22 gene adjuvant for enhancement of DNA vaccine against somatostatin in mice[J].Animal,2008,2(11):1569-1574.
[14] 王子馨,李坤,刘金朋,等.PCV2ORF2基因与猪IL-18基因共表达DNA疫苗的免疫原性[J].中国兽医学报,2013,33(4):532-537. WANG Z X,LI K,LIU J P,et al.Enhancement of DNA vaccine potency against PCV2 by coexpression of PCV2ORF2 and IL-18 gene[J].Chinese Journal of Veterinary Science,2013,33(4):532-537.(in Chinese)
[15] QI X X,KE B X,FENG Q,et al.Construction and immunogenic studies of a mFc fusion receptor binding domain (RBD) of spike protein as a subunit vaccine against SARS-CoV-2 infection[J].Chemical Communi-cations,2020,56:8683-8686.
[16] 侯丽萍,耿建林,谷巍,等.维生素D₃对实验性自身免疫性甲状腺炎Th1/Th2细胞平衡和MCP-1/CCR2信号的影响[J].重庆医科大学学报,2020,网络首发. HOU L P,GENG J L,GU W,et al.Effects of vitamin D₃ on helper T cell1/helper T cell2 cell balance and monocyte chemoattractant protein-1/cell surface chemokine receptor 2 signal in experimental automimmune thyroiditis[J].Journal of Chongqing Medical Univerisity,2020,Epub ahead of print.(in Chinese)
[17] OKAMOTO Y,NISHIDA M.Cytokine balance in the pathogenesis of rheumatoid arthritis[J].Akugaku Zasshi-Journal of the Pharmaceutical Society of Japan,2001,121(2):131-138.
[18] MCBRIDE W T,ARMSTRONG M A,CROCKARD A D,et al.Cytokine balance and immunosuppressive changes at cardiac surgery-contrasting response between patients and isolated CPB circuits[J].British Journal of Anaesthesia,1995,75(6):724-733.
[19] 王明雪,殷杰,张霓霓,等.艾迪注射液对兔舌VX-2鳞癌模型化疗外周血中Th1/Th2平衡的影响[J].广州中医药大学学报,2020,37(4):699-705. WANG M X,YIN J,ZHANG N N,et al.Effect of Aidi injection on Th1/Th2 balance in peripheral blood of rabbit tongue VX-2 squamous cell carcinoma model with chemotherapy[J].Journal of Guangzhou Univer-sity of Traditional Chinese Medicine,2020,37(4):699-705.(in Chinese)
[20] LONG R G,PETERS J R,GRAHAMESMITH D G,et al.Effect of somatostatin on provoked flushing and plasma guthormones in the carcinoid-syndrome[J].Gut,1980,21(5):A464.
[21] SPIER A D,DE LECEA L.Cortistatin:A member of the somatostatin neuropeptide family with distinct physiological functions[J].Brain Research Reviews,2000,33(2-3):228-241.
[22] 舒邓群,茆达干,陈荣达,等.粒细胞-巨噬细胞集落刺激因子与生长抑素融合表达质粒对断奶仔猪生长及相关激素的影响[J].南京农业大学学报,2007,4:92-96. SHU D Q,MAO D G,CHEN R D,et al.Effect of fusion-expression plasmid contained GM-CSF and SS on growth and relative hormone of weaned piglets[J].Journal of Nanjing Agrichitural University,2007,4:92-96.(in Chinese)
[23] HAN Y G,LIANG A X,HAN L,et al.Efficacy and safety of an oral somatostatin DNA vaccine without antibiotic resistance gene in promoting growth of piglets[J].Scandinavian Journal of Immunology,2014,79(4):244-250.
[24] 祖卓鑫.生长抑素和皮质抑素双表达非抗性筛选DNA疫苗的构建和鉴定研究[D].武汉:华中农业大学,2018. ZU Z X.Construction and evaluation of co-expression DNA vaccine against somatostatin and cortistatin without antibiotic resistance gene[D].Wuhan:Huazhong Agricultural University,2018.(in Chinese)
[25] 周子超.生长抑素DNA疫苗C500(pVGS/2SS-asd)对于育肥猪的生产性试验研究[D].武汉:华中农业大学,2019. ZHOU Z C.A field experiment of somatostation DNA vaccine C500(pVGS/2SS-asd) in growing pigs[D].Wuhan:Huazhong Agricultural University,2019.(in Chinese)