Study On The Conjugation Of Capsular Polysaccharide Of Streptococcus Zooepidemicus And Streptococcus Suis Type 2 With Porcine Circovirus VLP

Streptococcus suis disease is one of the three major bacterial diseases in breeding farms,which brings huge economic losses to the breeding industry every year.At the same time,it is also a common zoonosis,which threatens human life and health.Polysaccharide-conjugated vaccine is a kind of vaccine with good immune effect at present.The research and development of streptococcal polysaccharide-conjugated vaccine will bring great advantages to the prevention and control of suis streptococcal disease.In order to prepare the capsular polysaccharide of Streptococcus,the extraction method of capsular polysaccharide of Streptococcus pestilis was explored by taking Streptococcus pestilis for example.First,the strains were resuscitated and cultured,the bacteria solution was inactivated with 0.3%formaldehyde,and the cationic active agent was added to separate the polysaccharides from the bacteria.The bacteria solution was concentrated 20 times by rotary evaporator,and then centrifugation was carried out to make the bacteria precipitate and separate the supernatant containing polysaccharides.The ethanol fractional sedimentation method was used to sedimentation most nucleic acids and proteins at 25%ethanol concentration（V/V）,and all polysaccharides were precipitated at 80%（V/V）or above concentration.The crude polysaccharides were obtained by collecting and precipitation.High temperature protein denaturation method and deoxycholate sodium deproteinization method were used to remove proteins,and large molecular substances were filtered through 0.22 m sterilizing membrane.Finally,100 k D ultrafiltration tube was used for repeated filtration for 3-5 times.The relationship between bacterial growth and polysaccharide content over time was measured by CTAB method,and the polysaccharide content was the highest between 12h and 16h.The results showed that the concentration of polysaccharide was 1.68 mg/m L,the content of protein was 0.54%,and the content of nucleic acid was 0.31%.The purified product was identified as streptococcus capsular polysaccharide by nuclear magnetic resonance spectroscopy and scanning electronmicroscopy.In this study,the capsular polysaccharide of Streptococcus pestilis was successfully prepared,and the capsular polysaccharide of Streptococcus type 2 could be prepared by this method,which provided the basis for the research and development of the conjugated vaccine of Streptococcus.In order to investigate the reactivity of the conjugates of capsular polysaccharides and circovirus VLP,the two serotypes of capsular polysaccharides were conjugated to VLP by EDAC cross-linking method.The cross-linking was identified by NMR spectroscopy and scanning electron microscopy,and the coupling ratio was 4:1 as measured by protein single precipitation method.ICR mice were used as the animal model of the experiment and divided into 11 groups,namely CP2+CPS+VLP conjugate,CP2+CPS+VLP conjugate+FA,CP2+CPS+VLP simple mixture,CP2+CPS+VLP simple mixture+FA,CP2+FA,CP2 alone,VLP+FA alone,CPS alone,CPS alone and normal saline control group.The mice were sub CUM multipoint immunized.Blood samples at 14,21,28 and 35d were collected for antibody detection.The antibody test results showed that no antibodies were produced in the control group and the group without complete Freund’s adjuvant,while partial antibodies were produced in the group with adjuvant.In general,the antibody effect in the Cp2+CPS+VLP conjugate+FA group was better than that in the other groups.In order to investigate the immunogenicity of capsular polysaccharide with VLP conjugate of circovirus,challenge test was conducted in mice inoculated with this vaccine.By the plate count method measured per milliliter bacteria liquid contained in the bacterial count of 3.5 x 10⁹ cfu,bacteria will contain different Numbers of bacteria by intraperitoneal injection of liquid in mice to observe the deaths of mice after 72 h,measured streptococcus suis type 2 to the department of ICR mice most of lethal dose not concentration of 3.5 x 10⁸ cfu,all minimal lethal dose of 3.5 x 10⁹ cfu,and further experiment,through improved karber method to calculate LD₅₀ to streptococcus suis type 2to 1.59 x 10⁹ cfu.Mice vaccinated six different types of vaccines containing contact international.like apply SSDS,35 d measuring antibodies after intraperitoneal injection of 5times the LD₅₀ dose of bacteria liquid,the results showed that the physiological saline control group and without complete freund’s adjuvant group did not produce antibody,both tapping poison after mice died,plus adjuvant group part of antibody production in mice,tapping the poison after mice live,and in some survival rate and the rate of positive of antibody of quite,showed that antibodies to streptococcus suis type 2 strains of mice have certain protection.It provides important experimental data for the research and development of streptococcal polysaccharide conjugate vaccine.