CN112263674A - Porcine type 3 streptococcus capsular polysaccharide subunit vaccine and preparation method and application thereof - Google Patents
Porcine type 3 streptococcus capsular polysaccharide subunit vaccine and preparation method and application thereof Download PDFInfo
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- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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Abstract
The invention discloses a porcine type 3 streptococcus capsular polysaccharide subunit vaccine, a preparation method and application thereof. The capsular polysaccharide subunit vaccine is prepared from porcine streptococcus group 3 capsular polysaccharide. According to the invention, the porcine streptococcus 3 is taken as a research object, capsular polysaccharide is extracted from the porcine streptococcus 3 and is combined with a complete Freund adjuvant carrier to obtain the capsular polysaccharide subunit vaccine with strong specificity and immunoprotection efficacy, and a foundation is laid for the immune prevention and treatment of the porcine streptococcus 3.
Description
Technical Field
The invention belongs to the technical field of biological medicines. More particularly, relates to a preparation method and application of a swine 3 type streptococcus capsular polysaccharide subunit vaccine.
Background
Streptococcus suis is an acute and febrile infectious disease which is common to human and livestock and can cause meningitis, arthritis and septicemia of pigs, thereby causing serious economic loss to the pig industry. According to the antigenic difference of capsular polysaccharide, the streptococcus suis is divided into 35 serotypes (types 1-34 and 1/2), wherein the separation rate of 1/2, 1, 2, 7 and 9 is high, the pathogenicity of different serotype strains is different, the clinical separation rate of streptococcus suis type 2 is the highest, and the research reports of other serotypes are less. However, in recent years, the incidence of 3-type streptococcus suis has increased, and the 3-type streptococcus suis becomes a regional dominant serotype strain.
The vaccine immunization is considered as the most economic and effective means for preventing and controlling the streptococcus suis, and the popularization and the application of the commercial vaccine play an important role in effectively preventing and controlling the epidemic situation of the streptococcus suis in China. At present, commercial streptococcus suis vaccines in China comprise: the vaccine comprises a swine septicemic streptococcicosis live vaccine (ST 171 strain), a swine streptococcicosis inactivated vaccine (Streptococcus equi subsp zooepidemicus + Streptococcus suis type 2), a combined inactivated vaccine of the swine streptococcicosis and the haemophilus parasuis (LT strain + MD0322 strain + SH0165 strain), a swine streptococcicosis propolis inactivated vaccine (Streptococcus equi subsp zooepidemicus + Streptococcus suis type 2), a swine streptococcicosis inactivated vaccine (Streptococcus equi subsp zooepidemicus + Streptococcus suis type 2+ Streptococcus suis type 7). The 6 vaccines (1 live vaccine and 5 inactivated vaccines) are sold in the market, and the 5 related strains are mainly type 2 streptococcus. Due to the lack of cross-protection between different serotype streptococcus suis strains, there is an urgent need to develop a type 3 streptococcus suis vaccine for swine epidemic prevention in swine farms.
Capsular polysaccharide is a protective antigen and a virulence factor of bacteria, is a surface antigen with the least change of bacterial structures, has better immunogenicity, is one of the most suitable target antigens for vaccine components, and has better effect in preparing vaccines by purified capsular polysaccharide.
Disclosure of Invention
The invention aims to provide a subunit vaccine of streptococcus group 3 capsular polysaccharide of pig, which is characterized by comprising a combination of streptococcus group 3 capsular polysaccharide and complete Freund's adjuvant carrier, wherein the ratio of streptococcus group 3 capsular polysaccharide to complete Freund's adjuvant is as follows: 1 mixing, and the content of the type 3 streptococcus capsular polysaccharide is 5 mg/mL.
The invention further discloses application of the porcine type 3 streptococcus capsular polysaccharide subunit vaccine in preventing diseases induced by the infection of the porcine type 3 streptococcus, and experimental results show that the vaccine can provide virus attacking protection of homologous strains, can induce organisms to generate high-titer antibody levels, and reduce the clinical morbidity of the live porcine type 3 streptococcus.
The invention is described in more detail below:
the porcine type 3 streptococcus capsular polysaccharide can be prepared by the following preparation process:
(1) inoculating 3-type streptococcus suis (strain isolated and identified in the laboratory) in a test tube filled with 5mL Tryptone Soybean Broth (TSB), placing in a constant-temperature culture shaker for 12h, and preparing seed liquid for expanded culture; the TSB liquid culture medium comprises the following components: tryptone 1.5% (g/100 ml, soybean peptone 0.5% (g/100 ml), sodium chloride 0.5% (g/100 ml) were prepared with distilled water, pH was adjusted to 7.2. + -. 0.2, and the product was sterilized with steam at 121 ℃ and used.
(2) Inoculating the seed bacterial liquid into a 500ml LTSB liquid culture medium, and carrying out shake culture in a constant-temperature culture shaker for 12-14 h;
(3) centrifuging at 4 deg.C to collect bacterial sludge, dissolving with PBS and sterilizing with high pressure steam, and collecting all supernatants;
(4) adding crystalline salt-free egg white lysozyme, culturing the suspension at 37 ℃ for 6-8 h, and centrifuging to remove precipitates;
(5) adding protease K into the lysate, reacting at 55 deg.C for 2 hr, and simultaneously adding CaCl with final concentration of 0.1M2Stirring for 1 h;
(6) filtering the obtained supernatant with 0.22 μm fiber membrane;
(7) heating the obtained liquid on an electric furnace for concentration, stopping heating when the liquid becomes turbid, and quickly transferring to a water bath kettle at room temperature or a refrigerator at 4 ℃ for cooling;
(8) adding absolute ethanol according to the volume ratio (V/V) to obtain a final concentration ratio of 30%, and standing in a refrigerator at 4 ℃ overnight;
(9) centrifuging to collect supernatant, adding anhydrous ethanol to make final concentration of 80% (V/V), sealing with sealing film, and standing in refrigerator at 4 deg.C overnight;
(10) centrifuging, removing all supernatant to obtain white precipitate, drying with freeze-drying machine, and collecting to obtain capsular polysaccharide.
According to the preparation method of the subunit vaccine provided by the invention, the capsular polysaccharide and the complete Freund's adjuvant are prepared according to the proportion of 1: 1, mixing and emulsifying;
the subunit vaccine of the type 3 streptococcus capsular polysaccharide of the pig provided by the invention can stimulate an organism to generate an antibody for resisting the type 3 streptococcus capsular polysaccharide after intramuscular injection immunization.
The physiological and biochemical characteristics of the swine 3 type streptococcus capsular polysaccharide subunit vaccine disclosed by the invention are as follows:
(1) the content of the 3-type streptococcus capsular polysaccharide is 5 mg/mL;
(2) the invention is oil emulsion type and intramuscular injection mode.
Aiming at the phenomenon that the type-3 streptococcus suis clinically presents an advantage epidemic trend in recent years, the invention mainly solves the problem that the existing commercial streptococcus suis vaccine lacks cross protection, mainly inspects the possibility that capsular polysaccharide is used as subunit vaccine, and has the main difficulties of screening strains for the vaccine and extracting and purifying the capsular polysaccharide.
The swine 3 type streptococcus capsular polysaccharide subunit vaccine disclosed by the invention has the positive effects that:
(1) the protective rate of the capsular polysaccharide subunit vaccine of the invention to 3-type streptococcus is 75%;
(2) the invention can induce organisms to generate high-titer IgG and IgM antibody levels;
(3) the invention can reduce the incidence of 3-type streptococcus of piglets by about 60%.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available. Wherein the porcine 3-type streptococcus is separated and identified by the laboratory and is preserved in the laboratory (the specific separation and identification method is detailed in the separation and identification of the porcine 3-type streptococcus and the research of biological characteristics, the development of animal medicine, 2020, 41 (9): 11-15).
Specific characteristics of porcine streptococcus type 3:
gray, translucent and smooth round microcolonies with beta hemolytic rings with regular edges; carry aboutSrtA、orf2、Gapdh、 abpb、SsnA、Lmb、znuA、gdhgdh、epf、mrpA virulence gene; the growth curve shows that the strain reaches a peak growth (log CFU = 9.4)) between 9h and 10 h; the result of a drug sensitivity test shows that the isolated strain is sensitive to amoxicillin, cephalosporins and the like and resistant to florfenicol, lincomycin and the like; has strong pathogenicity on mice,the number of bacteria used is 1X 109The lethality of CFU/mL strain reaches 100% within 12h after infection.
Example 1
Preparation of streptococcus suis 3 capsular polysaccharide
1. Extraction of crude sugar
Streptococcus type 3 was inoculated into TSB liquid medium, cultured to logarithmic phase, washed with PBS after centrifugation, suspended in PBS liquid, and collected at 121 ℃ under high pressure. Adding crystal salt-free egg white lysozyme, culturing the suspension at 37 deg.C for 6-8 hr, centrifuging to remove precipitate, adding proteinase K into the lysate, acting at 55 deg.C for 2 hr, and simultaneously adding CaCl with final concentration of 0.1M2And stirring for 1 hour. The resulting supernatant was filtered through a 0.22 μm fiber membrane. Heating the obtained liquid on an electric furnace for concentration, stopping heating when the liquid becomes turbid, and quickly transferring to a water bath kettle at room temperature or a refrigerator at 4 ℃ for cooling. Absolute ethanol was added at a volume ratio (V/V) to a final concentration of 30%, and the mixture was allowed to stand overnight in a refrigerator at 4 ℃. Centrifuging at 25000 Xg for 30min, discarding the white precipitate, collecting supernatant, adding anhydrous ethanol to reach a final concentration of 80% (V/V), sealing with sealing film, standing in refrigerator at 4 deg.C overnight, and centrifuging at 25000 Xg for 30 min. And (4) pouring out all the supernatant to obtain a white precipitate, drying the white precipitate by using a freeze-dryer and collecting the white precipitate.
200mL of TSB medium is used for culturing type 3 streptococcus, capsular polysaccharide is extracted by the method, and 0.0887g of white powdery crude capsular polysaccharide is obtained after drying by a freeze-dryer. Measuring the content of capsular polysaccharide by a phenol-sulfuric acid method, preparing standard products by using glucose with different concentrations, expressing OD values by using abscissa, drawing a standard curve by using ordinate as sample concentration, obtaining a linear equation of Y =0.0106x, measuring the absorbance of the polysaccharide to be measured at 490nm to be 0.3227 by using an ultraviolet spectrophotometer, and calculating the content of crude capsular polysaccharide to be 34.21%.
2. Capsular polysaccharide purification
The dried white substance was accurately weighed. 5mg of the white powder was dissolved in 5mL of double distilled water, and DNA and RNase were added in time at a concentration of 100 mg/mL and the reaction was continued overnight at 37 ℃. Sampling and diluting by 50 times, and measuring the nucleic acid content by using a nucleic acid protein instrument, wherein the nucleic acid content is as follows: 0.0986mg/mL, protein content 0.0951 mg/mL.
Accurately weighing and dissolving 5mg of dried capsular polysaccharide in 5mL of double distilled water, adding protease K, and detecting the protein content. Samples were diluted in a gradient and 20uL was added to the sample wells of a 96-well plate. 200uL of BCA working solution is added into each hole, and the mixture is placed for 15-30min at 37 ℃. The protein concentration was calculated from the standard curve by measuring A560nm with a microplate reader. After purification, the polysaccharide content was calculated to be 71.12% by absorbance at 490nm with an ultraviolet spectrophotometer.
Example 2
Preparation of swine 3 type streptococcus capsular polysaccharide subunit vaccine
The purified capsular polysaccharide obtained from example 1 was diluted to 100ug/ml with sterile PBS and mixed with complete freund's adjuvant at a ratio of 1: 1, mixing uniformly, and preparing the capsular polysaccharide subunit vaccine for subsequent immunization after aseptic inspection.
Example 3
Swine source type 3 streptococcus capsular polysaccharide vaccine immune protection rate test
40 mice were randomly divided into 4 groups, capsular polysaccharide subunit vaccine test group, commercial vaccine (type 2+ 7) positive control group, challenge control group, blank control group. 0.2mL of capsular polysaccharide subunit vaccine is injected into a mouse muscle of a capsular polysaccharide subunit vaccine test group, 0.2mL of streptococcus group 2 and 7 general vaccine is injected into a positive control mouse muscle of a commercial vaccine (type 2+ 7), and 0.2mL of PBS solution is injected into a mouse muscle of each of an offending control group and a blank control group for blank immunization. At intervals of 14d, 1 booster immunization was followed by challenge with an intraperitoneal injection of a lethal dose of Streptococcus suis type 3. During the animal experiment, observing the mental state of the mice, recording the death condition of each group of mice, and calculating the protection rate; the protective rate of the capsular polysaccharide vaccine to 3-type streptococcus is 75%, the cross-protection rate of commercial vaccines (2 +7 type) to 3-type streptococcus is 37.5%, and the mortality rate of a blank challenge control group is 100%.
Example 4
Porcine streptococcus group 3 capsular polysaccharide vaccine antibody titer test
The detection method comprises the following specific steps: pre-coating specific anti-mouse IgG and IgM antibodies on an ELISA plate with high affinity, detecting the mouse IgG and IgM antibodies by a double-antibody sandwich enzyme-linked immunosorbent assay technology, and adding a standard substance and a sample to be detected into a hole of the ELISA plate for incubation. Two-step dilution is recommended. In the first step, 990. mu.L of 1 × detection buffer was added to 10. mu.L of the sample. In the second step, 10. mu.L of the mixed sample diluted in the first step was added to 990. mu.L of 1 Xdetection buffer. Firstly, washing the enzyme label plate for 1 time by using 300 mu L of 1 multiplied washing liquid and drying the washing liquid on filter paper, and pouring the washing liquid after soaking the enzyme label plate for 30 s, wherein the aim of the step is to balance the enzyme label plate; sequentially adding the standard substances diluted in multiple proportions in advance into the enzyme-labeled holes from top to bottom according to the concentration of the standard substances; then adding the serum of the mouse to be detected into the blank hole, after incubating for 2h, discarding the liquid in the hole, washing 5 times by using 1 Xwashing liquid, thoroughly washing away the unbound substances, then adding 100 mu L of detection antibody, and placing into a constant temperature incubator at 37 ℃ for incubation. After pouring out the liquid in the hole, washing the liquid for 5 times by using 1 Xwashing liquid again and patting the liquid on filter paper, adding 100 mu L of chromogenic substrate TMB into each hole, putting the hole into an incubator to be protected from light for reaction, and then adding a stop solution to stop the reaction. The absorbance was immediately measured using a microplate reader at a wavelength of 450 nm. The concentrations of IgG and IgM in mouse serum were calculated according to the calculation methods in the specification.
And drawing a standard curve by taking the IgG and IgM concentrations as abscissa and the OD value as ordinate, and respectively obtaining an IgG linear equation of y =0.0352 x-0.0208. The IgM linear equation is y =0.0467 x-0.0332. The contents of IgG and IgM in the mouse serum were calculated to be 0.1912. + -. 0.01458mg/mL and 0.8211. + -. 0.07718mg/mL, respectively. Through SPSS analysis, inp<And the 0.05 confidence interval shows that the content of IgG and IgM antibodies in the experimental group is obviously different from that in other groups, which indicates that the body can be induced to generate high titer antibody level.
Example 5
The pig farm application of the swine source type 3 streptococcus capsular polysaccharide vaccine is as follows: aiming at a certain pig farm with 3-type streptococcus positive, 60 weaned piglets of 4-5 weeks are selected, and 30 weaned piglets are selected respectively for an experimental group and a control group, wherein the experimental group is injected with 2mL of the product of the invention through muscle, the blank control is injected with physiological saline, the test period is 45 days, the clinical performance of the piglets is observed, and dead piglets or synovial fluid bacteria are separated. And (4) conclusion: the morbidity of the blank control group is 15 percent, the morbidity of the experimental group is 5 percent, and compared with the blank control group, the invention can reduce the morbidity of the 3-type streptococcus of the piglets by 60 percent.
Claims (2)
1. A subunit vaccine of Streptococcus group 3 capsular polysaccharide of porcine origin, characterised in that it comprises a combination of Streptococcus group 3 capsular polysaccharide and complete Freund's adjuvant carrier, wherein the ratio of Streptococcus group 3 capsular polysaccharide and complete Freund's adjuvant is in the range of 1: 1 mixing, and the content of the type 3 streptococcus capsular polysaccharide is 5 mg/mL.
2. The swine streptococcus group 3 capsular polysaccharide subunit vaccine of claim 1 is used for preventing diseases induced by streptococcus group 3 infection of swine.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1639569A (en) * | 2002-05-07 | 2005-07-13 | 坎莫森特里克斯公司 | Methods and compositions for inducing an immune response |
| CN102294026A (en) * | 2010-11-17 | 2011-12-28 | 赤峰博恩药业有限公司 | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1639569A (en) * | 2002-05-07 | 2005-07-13 | 坎莫森特里克斯公司 | Methods and compositions for inducing an immune response |
| CN102294026A (en) * | 2010-11-17 | 2011-12-28 | 赤峰博恩药业有限公司 | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| S D ELLIOTT ET AL.: "Streptococcal infection in young pigs.V.An immunogenic polysaccharide from Streptococcus suis type 2 with particular reference to vaccination against streptococcal meningitis in pigs", 《J HYG(LOND)》 * |
| 姜轩等: "天津地区猪7型链球菌的分离鉴定与小鼠致病性研究", 《黑龙江畜牧兽医》 * |
| 张奥等: "猪源3型链球菌分离鉴定及生物学特性研究", 《动物医学进展》 * |
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Application publication date: 20210126 |