| Streptococcus suis disease is one of the common bacterial diseases in pig farms,and type 2 is the most clinically isolated,most toxic and important pathogenic bacteria that threaten public health safety.In order to select the experimental strain with typical morphology and abundant capsule and understand its growth characteristics.The growth curve was determined by the method of biochemical and molecular biological identification,gram staining and capsular staining.The results showed that the three strains were Streptococcus suis2(SS2).By NCBI comparative analysis,the homology of the three strains was99.69% with a rich capsule,and the strain in TSB medium,0?2 h was the retardation period,2?5 h was the logarithmic period,5?9 h was the stable period,and 9 h was the decline period.The identification and understanding of the experimental strains provide important reference for the next step of mass culture and extraction of polysaccharide capsular.In order to improve the capsular polysaccharide(capsular polysaccharide,CP)the process of extraction and purification,is advantageous to the linear industrialized mass production.Eventually establish the capsular polysaccharide purification process that will select test strains bacteria was 18 h centrifugal take static culture mud,suspended in 0.1 M glycine buffer and adding lysozyme concentration to 1 mg/m L,magnetic stirring after 8 h centrifugal supernatant,20% ethanol precipitation to the nucleic acid,centrifugal supernatant,using 80% ethanol precipitation coarse polysaccharide.After the dissolution of the crude polysaccharide,PH was adjusted to below 5.0,Sodium deoxycholate(DOC)concentration was added to 0.5% to precipitate the deproteinization,and 0.22 m of bactericidal membrane was used to filter excessive molecular substances.Finally,100 KD ultrafiltration tube was used for repeated filtration and washing for 3-5 times.The purified polysaccharide group was divided into streptococcus suis type 2 capsular polysaccharide(CP2)by nuclear magnetic resonance spectroscopy(NMR).The results showed that the concentration of polysaccharide was 0.28 mg/m L,protein content accounted for 1.8%,and nucleic acid content was less than 1%.The optimized CP2 extraction and purification process in this study requires no high equipment investment,low cost and simple process,and also conforms to various indexes of polysaccharide capsular vaccine.It has laid a foundation for CP2 vaccine to go to clinical and commercial application.In order to evaluate the immune effects of different combinations of CP2 extraction and purification,the experiment first explored the use of glutaraldehyde and formaldehyde as cross-linking agents to co-precipitate Bovine serum albumin(BSA)and streptococcus suis type 2 capsular polysaccharide(CP2)with the optimal concentration of BSA of 0.5 mg/m L,the optimal content of glutaraldehyde of 4.8%and the optimal content of formaldehyde of 16.7%.ICR mice were selected as experimental animal models,and were divided into 6 groups: pure CP2,CP2 mixed with incomplete adjuvant(FIA),CP2 mixed with BSA,formaldehyde-crosslinked CP2,BSA,glutaraldehyde crosslinked CP2,BSA,normal saline negative control group,and subcutaneous immune mice.Blood was collected at the 14 th day of inoculation,and antibodies in serum of each group were monitored by indirect enzyme-linked immunosorbent assay.Antibody detection results showed that:CP2+IFA,CP2+BSA and formaldehyde cross-linked CP2 and BSA group began to produce antibodies on day 21,and the formaldehyde group had the highest antibody titer and the best immune effect.Important experimental data were provided for the research and development of CP2 vaccine... |
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