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Streptococcus suis type 2 three-component subunit vaccine and use

A subunit vaccine, Streptococcus suis technology, applied in the field of three-component subunit vaccine of Streptococcus suis type 2 and its preparation, can solve the problem of inability to clear bacteria of recessive infection, inability to clear type 2 Streptococcus suis, and the inability to exist anti-bacteria Drug particles and other issues

Active Publication Date: 2009-04-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inactivated vaccines have good immune protection against homologous Streptococcus suis, but due to the large differences in the virulence gene phenotypes of different strains, the protection against heterologous strains is not good; live vaccines can protect pigs, However, it cannot eliminate the type 2 Streptococcus suis colonized in the tonsils or joints, nor can it eliminate the bacteria in the tonsils of recessively infected pigs; live vector vaccines are mainly one of the main directions of current and future vaccine research and development, and the vaccines have both conventional The advantages of live vaccines and inactivated vaccines, but according to the regulations of the US Food and Drug Administration (Food and drug administration ration, FDA), drug-resistant plasmids cannot exist in live vaccines, and antibiotics cannot be used in humans and animals. To maintain the stability of the recombinant plasmid; the subunit vaccine has the advantages of high immune efficacy of the live vaccine and good safety of the inactivated vaccine, but the antigenic component of the subunit vaccine is single, and its protective effect on other serotypes is limited
The Streptococcus recombinant subunit vaccine developed by Fan Hongjie et al. can provide better protection against Streptococcus equi subsp. zooepidemicus and Streptococcus suis type 2. Streptococcus suis type also accounts for a certain proportion, while the proportion of Streptococcus equi subspecies zooepidemicus is getting smaller and smaller

Method used

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  • Streptococcus suis type 2 three-component subunit vaccine and use
  • Streptococcus suis type 2 three-component subunit vaccine and use
  • Streptococcus suis type 2 three-component subunit vaccine and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Cloning and expression of three kinds of Streptococcus suis type 2 antigenic proteins

[0030] one material

[0031] 1) Plasmids and strains

[0032] The plasmid pET-28a (+) that the present invention adopts is purchased from Noagen company (this pET-28a (+) plasmid map is as follows figure 1 Shown) Competent Escherichia coli BL21 (DE3) was purchased from Wuhan Life Technology Co., Ltd., Hubei Province, China.

[0033] The bacterial strain used in the present invention is the Streptococcus suis type 2 CVCC606 bacterial strain purchased from the China Veterinary Drug Control Institute in Beijing, China, and belongs to a commercial bacterial strain.

[0034] 2) The accession number of the gene derived from Streptococcus suis type 2, the position of the gene sequence in the genome of Streptococcus strains can be found in the appendix of the instructions figure 1 mentioned.

[0035] 3) Main reagents and buffer (Buffer)

[0036]Sodium chloride, EDTA disodium ...

Embodiment 2

[0124] Example 2: Characteristic Analysis of Recombinant Antigen Protein

[0125] 1. Western-blot analysis of the antigenicity of the recombinant protein

[0126] The above-mentioned purified recombinant antigen proteins 0197, 1036N and enolase were subjected to SDS-PAGE electrophoresis respectively. Proceed as follows:

[0127] 1) Transfer: Cut out 6 pieces of Whatman 3M filter paper and 1 piece of nitrocellulose membrane (NC membrane). The size of the filter paper and membrane should be exactly the same as the size of the gel or slightly smaller than the size of the gel. Mark the corner of the filter membrane with a pencil. Ensure the relative direction of the membrane and gel after transfer; soak the nitrocellulose membrane in purified water for 5 minutes; add a small amount of transfer buffer to another shallow tray, and soak 6 pieces of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank as follows: lay the base (anode) of the graphite electrod...

Embodiment 3

[0143] Embodiment 3: Recombinant antigenic protein immune protection test

[0144] Expression and purification of recombinant antigens

[0145] The expression strains containing pET-28a-0197, pET-28a-1036N, and pET-28a-enolase plasmids were inoculated in 3 mL LB liquid medium containing 0.25 μg / ml, respectively, and cultured on a shaker at 37°C. Take 100 μL of the cultured bacterial liquid and inoculate it into 10 mL of fresh LB liquid medium containing 2.5 μg kanamycin, culture it with shaking at 37°C for about 3 hours, until OD 600 When it reaches 0.6-1.0, add IPTG to a final concentration of 0.8mmol / L, continue to cultivate for 3h, and then collect the bacteria.

[0146] The recombinant antigen-expressing cells collected above were resuspended with Binding buffer (20mM Tris-HCl pH7.9, 5mMlmidazole, 0.5MNaCl), ultrasonically disrupted, centrifuged at 12,000g at 4°C for 15min, and the supernatant was taken for loading. Use Binding buffer and Washing buffer (20mM Tris-HCl pH...

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Abstract

The present invention belongs to the technical field of murrain vaccine preparation, and in particular relates to a Streptococcus suis type 2 trimaceral subunit vaccine and a preparation method thereof. The key technology is that recombinant Escherichia coli such as Escherichia coli BL21 / pET-28a-1036N, Escherichia coli BL21 / pET-28a-0197 and Escherichia coli BL21 / pET-28a-enolase is prepared, which can excrete and express Streptococcus suis type 2 antigen albumen 1036N,0197 and enolase, and which is preserved as CCTCC NO M208147, CCTCC NO M208146 and CCTCC NO M208148 in China Center for Type Culture Collection respectively. The present invention also discloses a preparation method and a use suitable for the trimaceral subunit vaccine of Streptococcus suis type 2.

Description

technical field [0001] The invention relates to the technical field of preparation of livestock infectious disease vaccines. It specifically relates to a three-component subunit vaccine of Streptococcus suis type 2 and its preparation method and application. The invention relates to the cloning, expression, functional verification and application of three antigenic protein genes based on Streptococcus suis type 2. The antigenic protein expressed by the gene can improve the ability of pigs to resist Streptococcus suis type 2. Background technique [0002] Streptococcus suis (S.suis) is the main pathogen that causes streptococcus suis. According to the capsular polysaccharide (CPS) on the surface of the bacteria, S.suis can be divided into 35 serotypes, namely type 1 / 2 and Types 1 to 34, but recently it has been suggested that types 32 and 34 should be attributed to Streptococcus orisratti instead of Streptococcus suis, among which Streptococcus suis type 2 is the most virul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/00A61K39/09A61P31/04C12R1/19
Inventor 金梅林李冉张安定陈焕春王雅康超陈博
Owner HUAZHONG AGRI UNIV
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