Preparation Of Live Recombinant Lactococcus Lactis Vaccine Expressing Sip Protein Of Streptococcus Agalactiae Isolated From Tilapia(Oreochromis Niloticus)and Immunogenicity Analysis

Tilapia has been widely bred in southern China and has become one of the major aquatic species in China.It has high economic value and has greatly promoted China's import and export trade.In 2009,a large-scale outbreak of tilapia infected Streptococcus agalactiae in major areas of southern China,which severely threatened the healthy development of tilapia aquaculture.At present,there is no effective prevention and treatment method,while the main prevention and control measures are antibiotics.Antibiotics are likely to cause deterioration of the breeding environment because of their drug residues.The vaccine has the advantages of safety,high efficiency and no residue.In order to carrying out healthy aquaculture,vaccine controlling will be a future aquaculture disease.At present,vaccines have inactivated vaccines,attenuated vaccines,DNA vaccines,and genetic engineering subunit vaccines.As the existing vaccines have various limitations,new vaccines have become the main trend of prevention and controlling S.agalactiae.In order to developping a vaccine which is easy to immunize and has a better immunity,a shuttle plasmid that expresses the surface immunogenic protein?Sip?of S.agalactiae was constructed and recombinant expression was prepared.The live lactic acid bacteria vaccine was used for immunogenicity study by oral administration.The main research contents and results are as follows:First part:construction and prokaryotic expression of Sip gene shuttle expression plasmid of Streptococcus agalactiae in Nile tilapiaIn order to develop vaccines for tilapia-resistant streptococcus disease with better immune effect and brief operation,this study constructed shuttle plasmids pNZ8124-sip and pNZ8148-sip that recombinantly express Sip proteins of S.agalactiae,in which the pNZ8124-sip plasmid carries the secreted protein gene Usp45.The signal peptide sequence was confirmed by digestion and sequencing,then transfected into Lactococcus lactis NZ9000 to obtain L.lactis inducing recombinant expression of Sip protein.The SDS-PAGE electrophoresis was used to explore the optimal condition to obtain the maximum expression.The target protein was purified by nickel column and detected by Western blot.The results showed that the constructed recombinant L.lactis can induce the expression of a specific protein with a size of 48 KDa by nisin,and the non-secreted expressed protein is45KDa,which is consistent with the size of the target protein.SDS-PAGE electrophoresis showed that the recombinant protein exists mainly in the form of soluble proteins and inclusion bodies.Among them,the soluble protein concentration reached 7.65 mg/mL;the optimal nisin concentration of inducing expression was 100ng/mL and the time was 6 h;Western blotting showed that the induced protein could specifically bind to mouse anti-His tag antibody.This study successfully constructed the Sip protein of secreting L.lactis NZ9000-pNZ8124-sip and the non-secreting strain NZ9000-pNZ8148-sip of S.agalactiae,which induced the expression of a large amount of soluble Sip protein.The preparation of a live vaccine vector vaccine for Sip protein provided the basis for the next step of immunogenicity assay.Second part:immunogenicity analysis of Sip Protein lactic acid vaccine in Nile tilapiaIn order to analyze the immunogenicity of the live vector Sip protein lactic acid vaccine,the NZ9000-pNZ8124-sip and NZ9000-pNZ8148-sip recombinant strains were obtained at the optimal induction condition of nisin,which orally immunized Nile tilapia with the dose of 2.24×10⁹?low concentration group?,2.24×10¹⁰?medium concentration group?and 2.24×10¹¹ CFU/mL?high concentrati on group?100?L per tail.NZ9000-pNZ8124,NZ9000-pNZ8148 and NZ9000 strains and PBS solution were given orally separately,with the oral doses of control group was 2.24×10¹⁰ CFU/mL.The second immunization was performed after a week the first immunizations.After the first immunization,3 fishes were randomly selected from each group on the 1st,2nd,4th,8th,16th,and 21st days.Blood was collected from the tail vein,then the serum was separated for next expeiment.On the 1st,2nd,4th,8th and 16th days 3 fishes were randomly selected to collect thymus,liver,spleen and intestine tissues for detecting the relative expression levels of immune-related genes IgT,IgM,CD8a,and C3 by fluorescent quantitative PCR;the activity of superoxide dismutase?SOD?,alkaline phosphatase?AKP?and lysozyme were determined by the kit method;the changes in serum antibody levels used indirect ELISA to analyze.On the 21st day after the first immunization,S.agalactiae WC1535 was used for intraperitoneal injection of artificial infection.The concentration used for the challenge was 2.25×10 ⁷ CFU/mL?LD50?100?L per tail.calculating the relative immunity survival rate?RPS?of each group.The RPS was obtained infecting S.agalactiae by artificial intraperitoneal injection.After oral immunization with NZ9000-pNZ8124-sip and NZ9000-pNZ8148-sip(2.24×10¹⁰CFU/mL),the result of the relative expression levels about IgT,IgM,CD8a,and C3genes were detected in thymus,liver,spleen and intestinal tissues showed an upward trend during the first immunization,a downward trend with time going on,and a trend of upward adjustment after the second immunization.The activity of lysozyme,SOD and AKP in tilapia serum were significantly higher than PBS group on 21st day.The serum antibody of NZ9000,NZ9000-8124,NZ9000-8148,NZ9000-8124-sip(2.24×10¹¹ CFU/mL)and NZ9000-8128-sip(2.24×10¹¹ CFU/mL)were not significantly higher than PBS group;the antibody levels about medium concentrations of NZ9000-8124-sip(2.24×10¹⁰ CFU/mL)and low concentrations of NZ9000-8124-sip?2.24×10⁹ CFU/mL?could be significantly increase and showed a trend of increasing first and then decreasing with time going on,which was 16 d and 4 d at the peak respectively.the antibody concentration in the medium concentration group was higher than in the low concentration group;double immunities were significantly improved in the tilapia,and the highest immune protection rate was 41.0%in the medium concentration group.The serum antibody levels of the medium concentrations of NZ9000-8148-sip(2.24×10¹⁰ CFU/mL)and high concentrations of NZ9000-8148-sip?2.24×10¹11 CFU/mL?significantly increased,and showed a trend of increasing first and then decreasing with time and both peaked at the 21st day,while the antibody level in the medium concentration group was higher than that in the low concentration group.The highest immune protection rate in the medium-dose group was 61.6%.The effect of the non-secreted live vector vaccine was better than the secretory type in analyzing the overall immune effect.This study can lay a foundation for oral vaccine of O.niloticus against S.agalactiae and has a broad prospect of application.