Study Of Drug Delivery System Development To Anti Micropterus Salmoides Rhabdovirus

Micropterus salmoides rhabdovirus（MSRV）is a virus that seriously harms the healthy growth of largemouth bass.The outbreak of the virus will bring serious economic losses to my country’s largemouth bass aquaculture every year.As we all know,immune prevention and control are effective prevention and control methods of viral diseases,and it is also an important measure to ensure the breeding results of largemouth bass.In the aquaculture industry,injection,bathing,and oral administration are the three conventional immunization methods.Although injection immunization has the best effect,the high labour intensity and severe emergency response of fish have become difficult problems in the promotion and application in production.Although immersion and oral immunization can be scaled,factors such as the barrier of the fish body and the degradation of the intestine make their effectiveness greatly reduced.In the early stage of our laboratory,a series of progress has been made in the research of genetically engineered vaccines carried by inorganic carbon nanotubes,which provides a reference for the development of this research.Therefore,the biodegradable organic material of Bacterial Cellulose（BC）was used to prepare nanocellulose,and through the optimization of the preparation process,the construction of the nanocellulose-loaded Rhabdovirus G2 protein vaccine and the effect evaluation,to provide basic information for industrial application,yielding the following results:1.Preparation and study of functionalized Bacterial nanocelluloseFirst,the bacterial cellulose was broken,and 2,2,6,6-tetramethylpiperidine nitrogen-oxygen-oxide（TEMPO）was subjected to carboxylation modification to prepare the carboxylated bacterial cellulose.Using the controlled variable method,carboxylated bacterial cellulose was dosed proportionally to different proportions of sulfuric acid（55%,65%and 70%）.At50°C,under the same concentration of sulfuric acid treatment,acidification was performed for 0and 120 min,respectively.Characterized by atomic force microscopy,carboxylated bacterial nanocelluloses with different average sizes（1000,600,350 nm）were prepared.Bacterial nanocelluloses of different sizes were labeled with rhodamine isothiocyanate,incubated with zebrafish embryonic cells（ZF-4）at concentrations of 15,30,and 60 mg L-¹,respectively,and screened by flow cytometry Analysis,bacterial nanocellulose with an average size of 350 nm was more easily taken up by cells.The adults and larvae of zebrafish were soaked with 30 mg L-¹rhodamine isothiocyanate-labeled bacterial nanocellulose,respectively.After 5 h,the adults and larvae were collected,and tissue sections were made and detected by laser confocal microscopy.The enrichment of bacterial nanocellulose in various tissues and organs in zebrafish,and the distribution of bacterial nanocellulose in larvae.The experimental results show that bacterial nanocellulose has good penetration performance.ZF-4 cells were co-incubated with bacterial nanocellulose（350 nm）at 15,30,and 60 mg L-¹,respectively;zebrafish were immersed in zebrafish at concentrations of 100 and 500 mg L-¹.The experimental results showed that.The test results showed that compared with the control group,bacterial nanocellulose did not have a significant effect on cell viability（P<0.05）;the enzyme activity levels in zebrafish after immersion for 72 h were significantly different from those in the control group（P<0.01）,but no zebrafish deaths were recorded in the next 30 days.2.Preparation BNCs-G2 vaccine systemThe predominant antigenic epitope of the largemouth rhabdovirus（glycoprotein,G）was screened in the laboratory’s preliminary research,and the test results showed that the G2 epitope has good immunogenicity.In this study,the prokaryotic expression system was used to ferment the G2 protein,and the fermented protein was purified.The purified protein was verified by protein gel and WB,and the G2 protein was successfully expressed.The carboxylated bacterial nanocellulose prepared in the previous section was used to link with the G2 protein through an esterification reaction.Using atomic force microscopy to characterize the bacterial nanocellulose before and after the protein was attached,the experimental results showed that after the bacterial nanocellulose was attached to the protein,the diameter of the nanofiber was larger than that of the nanofiber that was not attached to the protein.The concentration of bacterial nanocellulose-loaded protein was measured,and the antigenic protein accounted for about 24.3%of the nano-loaded vaccine system.Using 100 mg L-¹ bacterial nanocellulose-loaded G2 protein vaccine system（BNCs-G2）to bathe largemouth bass,after 30 days,compared with the control group,the largemouth bass in the vaccine bathing group did not die,proving that Vaccine systems have a proven safety profile.3.Study of BNCs-G2 vaccine systemThe Largemouth bass（1±0.2 g）were randomly and equally divided into eight groups,they bathed with BNCs-G2（10,20,40 mg L-¹）,naked G2（10,20,40 mg L-¹）,PBS and BNCs.The protective effects were assessed by analyzing MSRV G proteins-specific serum antibodies levels and the expression of four immune-related genes（Ig M,IL-12,TCR,and TNF-α）.Results showed that serum antibodies levels and the expression of immune-related genes were significantly increased for BNCs-G bath-vaccinated groups（40 mg L-¹）,compared with PBS and the naked G protein groups.After bath immunization for four weeks,fish were challenged with a lethal dose of MSRV.The survival rates for control group（PBS）,BNCs group(40 mg L^-1),the naked G protein groups(40 mg L^-1)and three BNCs-G groups(10 mg L^-1,20 mg L^-1and 40 mg L^-1)were 0%,0%,30.0%,16.7%,46.7%and 66.7%,respectively.These results indicated that BNCs can be a plausible candidate for the vaccine carrier system and the BNC loaded glycoprotein showed good immunogenicity.All in all,the BNC can be a great carrier of vaccines and drugs,which can improve the vaccines and drugs concentration in largemouth bass,and improve the survival rate of largemouth bass.This study proves the evidence of BNC can as the nano carrier system.And it has significant means to Chinese aquaculture development.