转基因vp28蓝藻口服疫苗半数有效量测定及其对斑马鱼的安全评价
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S 942.3

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国家重点研发计划(2019YFC0312604);上海市农委科技兴农项目[沪农科推字(2017)第1-13号];国家“八六三”高技术研究发展计划(2014AA093506);上海市大学生创新训练计划项目(S201910264057)


Determination of ED50 of transgenic cyanobacteria oral vaccine and safety evaluation on Danio rerio
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National Key Research and Development Project (2019YFC0312604); Shanghai Agriculture Science and Technology Innovation Project (2017, 1-13); National Marine 863 Project (2014AA093506); Student Innovation Training Project (S201910264057).

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    摘要:

    为了探究转基因蓝藻作为口服疫苗来防治白斑综合征病毒(WSSV)的效果,本研究将转基因vp28蓝藻口服疫苗投喂凡纳滨对虾幼苗,验证其剂量梯度抗WSSV的影响,并测定其半数有效剂量(ED50)。以该剂量为依据投喂斑马鱼,通过测定斑马鱼体内的酶学指标、观察组织切片及养殖水体中氮磷等的变化,来探究转基因vp28蓝藻口服疫苗对水生生物和水体环境造成的影响。研究结果显示,随着转基因蓝藻口服疫苗剂量的增加,凡纳滨对虾幼苗抗WSSV的能力随之增强,测得ED50为0.027 g。分别使用野生型和转基因型蓝藻配合饲料投喂斑马鱼15 d,投喂结束后其生长状态良好、游动正常;投喂前后身体颜色无明显差异,不同组间体长增长不显著。转基因组斑马鱼中过氧化氢酶活力稍低于空白组和野生型组,过氧化物酶活力随着投喂天数的增长而呈降低趋势;水质监测发现,总氮日均波动变化且变化幅度较小,总磷在投喂期间有所提升,15 d时恢复到初始投喂日的水平。投喂蓝藻的2组氨氮均要低于空白对照组,氨氮含量的提升对斑马鱼的抗氧化能力存在一定影响;组织切片发现,各组斑马鱼的肝脏、心脏组织细胞无明显差异。研究表明,转基因vp28蓝藻口服疫苗可有效增强凡纳滨对虾抗WSSV能力,且抗病能力随着口服疫苗剂量的升高而增强;投喂斑马鱼后,斑马鱼无明显的毒理效应,且对水质影响较小;转基因vp28蓝藻作为亚单位疫苗投放对水生生物的影响较小,可为后续产业规模应用提供基础。

    Abstract:

    Nowadays, in aquaculture, the popularity of antibiotics and the increase in the use of other drugs have made it easier for residual antibiotics and drugs to invade natural water bodies. The environmental effects of antibiotics in the ecosystem have caused more and more pollution to the water environment and threatened the survival of aquatic organisms. After strict regulation and control of the use of antibiotics, the use of genetically modified vaccines that can replace antibiotics has become more and more common. Cyanobacteria is a natural bait for shrimp seedlings. Directly feeding Litopenaeus vannamei with Synechococcus-expressedvp28 to control WSSV can play a role of “medicinal and food homology”. However, the problem facing the current application of transgenic cyanobacteria as a subunit vaccine is the safety of its environmental release. Danio rerio is one of the most commonly used model creatures. In the previous Synechococcus-expressedvp28 oral vaccine efficacy experiment, the feasibility of the oral vaccine was verified, showing that its efficacy and safety can be trusted. This environmental release experiment was built on this basis. In this study, Synechococcus-expressedvp28 were administered to L. vannamei to verify the effect of its dose gradient against WSSV and to determine its half effective dose (ED50). After feeding D. rerio on the basis of this ED50, the effects of the Synechococcus-expressed vp28 on aquatic organisms and water environment were explored by measuring the enzymatic indicators in D. rerio, observing the tissue sections, and the changes in nitrogen and phosphorus in the aquaculture water. The results of the study showed that with the increase of the dose of Synechococcus-expressedvp28 oral vaccine, the anti-WSSV efficacy of L. vannamei also increased and the measured ED50 was 0.027 g. Feeding D. rerio by freeze-dried wild-type and transgenic Synechococcus with the shelled-out Artemia for 15 days, the body length and color of D. rerio were not significantly different. The CAT in the transgenic group was significantly lower than that blank and wild-type groups. There was no significant difference in SOD and POD between wild-type and transgenic groups. There was no significant difference in the liver and heart of D. rerio through the tissue section. The ammonia nitrogen in wild-type and transgenic groups was lower than that in the blank group, and there was no significant difference between total nitrogen and total phosphorus. The results of this study further showed that the Synechococcus-expressed vp28 can effectively enhance the anti-WSSV ability of L.vannamei, and as its dose increases, the disease resistance has a more significant expression. In the current experimental results, there is no obvious toxicological effect on D. rerio, and after feeding with Synechococcus-expressed vp28, it does not show a significant effect on the quality of aquaculture water. This shows that this subunit vaccine has less impact on aquatic organisms, and can provide a basis and more possibilities for subsequent industrial-scale applications and development.

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徐杨,谢京昆,李赟卉,苏永政,何培民,贾睿.转基因vp28蓝藻口服疫苗半数有效量测定及其对斑马鱼的安全评价[J].水产学报,2021,45(2):255~264

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  • 收稿日期:2020-02-04
  • 最后修改日期:2020-06-19
  • 录用日期:2020-07-02
  • 在线发布日期: 2021-01-30
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