| Tilapia is an important economic fish,and it accounts for a large proportion of the world’s aquaculture production.Streptococcus agalactiae is an common pathogenic bacteria.Many hosts such as humans,cattle,and fish are susceptible to this pathogenic bacteria.For many years,Streptococcus agalactiae has seriously threatened the development of tilapia aquaculture.Because of their green,biosafe,and high-efficiency,vaccines have been becoming one of the best choice for the prevention and control of fish diseases.The studiess focused on fish vaccines are very popular in recent years,while only fewer types of vaccines are commercially available.Because fish live in an open water environment,the cost of injection immunization is high,so oral immunization is the most suitable method of immunization for fish.Antigens can be easily damaged by Fish gastrointestinal digestive enzymes.Therefore,the development of adjuvants that can improve the stability and absorption rate of vaccines is of great significance for oral fish vaccines.Mesoporous silica nanoparticles（MSN）have become carriers of many antigens because of their strong safety,good stability,and high loading rate.They are currently a popular type of material in the medical field.Hydroxypropyl Methyl Cellulose Phthalate（HP55）has been used as coating for many vaccines due to its pH response.It is promising to take advantage of these raw materials to produce new adjuvants with excellent characteristics for fish nano-vaccine.The existing research shows that the surface immunogenic protein（SIP）of Streptococcus agalactiae is an effective candidate protein for subunit vaccines.Therefore,this study chose to use SIP as the antigen,and use biological information technology and genetic engineering technology to analyze,express and purify SIP protein.The nano-material encapsulated SIP is prepared by chemical synthesis,and subsequently the performance of nano-vaccine is tested by simulating gastrointestinal environment and MTT colorimetry.The immune protection effect of SIP encapsulated by nano-materials on tilapia are evaluated by combining serum detection,intestinal immunohistochemistry,tissue immune factor detection and other methods.The specific research content and results are as follows:1.Using double enzyme digestion and sequencing,it was verified that the SIP gene was 1392 bp in length,encoding 463 amino acids,with a relative molecular weight of48.64 k Da.Recombinant bacteria was used to induce soluble expression by IPTG,and then purified by affinity chromatography to obtain a large amount of SIP protein.Molecular sieve experiment was used to analyze whether SIP protein is dimerized or polymerized,and mice were immunized with purified SIP protein.The indirect ELISA assay indicated that the mouse antiserum with a titer of 1:128000.The mouse serum was purified by protein A sepharose.The mouse polyclonal antibodies were obtained at a concentration of 3.72 mg/m L.2.Using gel-sol synthesis technology to prepare MSN material,after physical loading of SIP protein,HP55 was introduced into SIP-MSN encapsulation by double emulsion method to prepare SIP-MSN@HP55 vaccine,and the adhesive was used with fish The feed is mixed uniformly,and chitosan was added to make vaccine feed.The simulated artificial gastrointestinal fluid was used to analyze the pH response and sustained release effect of the SIP-MSN@HP55 vaccine.The results showed that the SIP-MSN@HP55 vaccine can protect the antigen protein well in the gastric fluidenvironment（pH 1.5）,and in the intestinal juice environment（pH 7.4）.)The antigen protein can be quickly released 15 minutes before the next step,and then the remaining SIP can be slowly released.The MTT assay showed that the cell survival rate of the SIP-MSN@HP55 vaccine at the test dose（120 μg/m L）was above 88.01%,which proved that the prepared nano-vaccine had good biological safety.3.After oral immunization of tilapia with vaccine feed,ELISA detected that the tilapia serum antibody titer of SIP-MSN@HP55 group reached 1:200 at 21 days,and the antibody level was significantly higher than SIP groups（P<0.05）.Intestinal immunohistochemistry obtained antigen uptake,and found that SIP can be absorbed by the intestinal lamina propria,and enter the body tissues through the intestinal blood circulation.RT-q PCR experiments were used to detect CD-4,CD-8,TNF-α,Ig M,IL-10,IFN-γ in the spleen,intestine,and kidney tissues of tilapia at 7,14,21,and 28 days after immunization.Changes in the expression levels of immune-related factors showed that the expression levels of immune factors were up-regulated to varying degrees,and various immune factors in the SIP-MSN@HP55 group were significantly up-regulated compared with other groups（P<0.05）.Finally,in the second week after the booster immunization,different concentrations of Streptococcus agalactiae Tn3 L were injected intraperitoneally with tilapia for a challenge test,and it was concluded that the relative protection rate of the SIP-MSN@HP55 group could reach more than 63.33%.In this study,nanomaterials were prepared to encapsulate SIP protein.The drug delivery system has the characteristics of controlled release of pH and good biocompatibility,which improves the efficiency of antigen utilization.In summary,we have developed an economical and applicable tilapia SIP-MSN@HP55 nano oral vaccine,which could stimulate the innate and adaptive immune response of tilapia to protect tilapia from infecting with Streptococcus agalactiae.Protection effect.This suggests that MSN is a potential fish vaccine delivery system,and can also provides reference value for the further research and development of fish nano-vaccine. |
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