Study On The ED₅₀ And Environmental Safety Evaluation Of Vp28-expressed Synechococcus Sp. PCC 7942 Oral Vaccine

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.White spot syndrome virus is one of the most serious viruses in the global shrimp farming industry.Previous studies have shown that the vp28-expressed Synechococcus sp.PCC 7942 can be used as bait for shrimp seedlings.Directly feeding Litopenaeus vannamei with Synechococcus-expressed vp28 to control WSSV can play a role in“medicinal and food homology”.In this study,vp28-expressed Synechococcus 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 based on this ED₅₀,the effects of the vp28-expressed Synechococcus 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.Compared the effects of exposure to different kinds of algae fluids of D.magna,analyzed its effects on the nervous system,antioxidant system and immune function enzyme activity,and analyze its m RNA transcription level,and use D.magna as a model organism to evaluate the biosafety of transgenic vp28-expressed Synechococcus.The main results are as follows:（1）The results of the study showed that with the increase of the dose of Synechococcus-expressed vp28 oral vaccine,the anti-WSSV efficacy of L.vannamei also increased and the measured ED₅₀was 0.027 g.（2）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.（3）Using Scenedesmus obliquus and wild-type,p RL489,vp28 Synechococcus sp.PCC 7942 to D.magna.According to the guidelines of OECD-202 and OECD-211,21-day chronic and 7-day acute experiments were performed.It was found that the limb structure,swimming behavior and feeding behavior of the D.magna were intact,and it was found that fed different species of algae or different substances of the same algae species would change the number of eggs laid and the total number of fetuses while changing the species of algae would increase its peeling rate;The enzyme activities of CAT and GST after feeding Synechococcus were significantly different from those of Scenedesmus obliquus,but there is no significant difference in ACh E activity.The difference of the algae transfected with vp28 gene was not significant,and the gene expression did not have a significant impact on the corresponding enzyme activity.The results of this study further showed that the vp28-expressed Synechococcus 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 D.magna,after feeding with vp28-expressed Synechococcus,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.