Streptococcus suis type 2 10-gene-deleted strain and application

A technology of Streptococcus suis, gene deletion, applied in bacteria, antibacterial drugs, veterinary vaccines, etc., can solve the problem of ineffective activation of the mucosal immune system, and achieve the effect of avoiding immunogenic damage, decreased virulence, and genetic stability

Active Publication Date: 2016-11-09
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many vaccine research and patent achievements have achieved immunization through injection, and the way of immunization through injection has certain limitations in activating the mucosal immu

Method used

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  • Streptococcus suis type 2 10-gene-deleted strain and application
  • Streptococcus suis type 2 10-gene-deleted strain and application
  • Streptococcus suis type 2 10-gene-deleted strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The acquisition of Streptococcus suis 10 gene deletion strain LSM110D10:

[0060] According to the complete genome GB|CP000407.1| of Streptococcus suis type 2 05ZYH33 strain published on GenBank, primers were designed to amplify the upstream homology arm and downstream homology arm of the gene to be deleted from the SC genome of Streptococcus suis type 2 wild strain, Construct a knockout vector for knockout of the target gene. The applicant constructs and screens the deletion strain by using a reverse selection marker through a two-step single-exchange homologous recombination method between the recombinant plasmid and the strain genome. This method can be used repeatedly to construct polygenic mutants without leaving any selection markers, see the related report of Dr. Daisuke Takamatsu (Daisuke Tetal, 2001, 45:101-113).

[0061] After the target gene is knocked out, design a pair of primers located on both sides of the gene and a pair of primers located inside the ge...

Embodiment 2

[0069] Characteristics of Streptococcus suis 10 gene deletion strain LSM110D10:

[0070] 1) Observing under the electron microscope the virulent strain SC of Streptococcus suis type 2 and the 10 gene deletion strain of Streptococcus suis LSM110D10, both of them formed normal bacterial morphology, cell wall and capsule, and there was no significant difference in the morphology of the two, Figure 6.

[0071] 2) Determination of drug resistance. The virulent strain SC of Streptococcus suis type 2 and the 10 gene deletion strain of Streptococcus suis LSM110D10 were respectively inoculated in 5 mL of TSB medium containing 10% newborn bovine serum, cultured to logarithmic phase at 37°C, and the concentration was adjusted to 1.2× 10 9 CFU / mL, take 100μL and evenly spread it on the MH+10% sheep blood plate, paste the penicillin E-test strip, carefully drive out the air bubbles, and take a reading after incubation at 37°C for 24 hours. The result is as follows: Figure 7 , Figure...

Embodiment 3

[0076] The safety of Streptococcus suis 10 gene deletion strain LSM110D10:

[0077] 1) Determination of hemolytic activity. The strain was inoculated in 5 mL of TSB medium containing 10% newborn bovine serum and cultivated to the logarithmic phase, and the concentration was adjusted to 5×10 7 CFU / mL, add 100 μL to 900 μL 5% fresh sheep red blood cells, add 100 μL PBS buffer to the negative control group, incubate at 37°C for 2 hours, centrifuge to get the supernatant, measure the absorbance at 570nm, the results are as follows Figure 9 , Figure 9 It is the hemolytic activity determination result of LSM110D10 of the present invention and the virulent strain SC of Streptococcus suis type 2, and a 3-deletion mutant strain without knocking out the hemolysin gene Sly is used as a control, obtained by Figure 9 It can be seen that the OD value of SC and 3 deletion can reach about 0.9, while the OD values ​​of LSM110D10 and the blank control group are both about 0.1, indicating t...

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Abstract

The invention discloses a streptococcus suis type 2 10 gene-deleted strain and application. The strain is obtained by knocking out 10 genes on the basis of a streptococcus suis type 2 virulent strain, and the preservation serial number is CCTCC NO: M 2015800. The virulence of the strain is significantly reduced compared with that of the parent strain, safety is high, immunogenicity is high, and immunized mice can be protected against the attack of the streptococcus suis type 2 virulent strain after being immunized in an injection or nasal drip or oral administration mode, which shows that the vaccine can effectively activate the mucosal immune system and systemic immunology of a host; meanwhile, a cross protection function on streptococcus suis type 2 virulent strains of different ST types can also be achieved. The vaccine is simple in preparation method and suitable for industrial production; meanwhile, the immunogenicity of the mutant strain is similar to that of a wild virulent strain, and thus the mutant strain can be applied to the development of rapid reagent kits as a standard substance or a whole-cell antigen.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering vaccines for livestock infectious diseases, and in particular relates to a type 2 10-gene deletion strain of Streptococcus suis and its application. Background technique [0002] Streptococcus suis is an important zoonotic pathogen, Streptococcus, Gram-positive, facultative anaerobic, can produce α or β hemolysis on blood plate, the bacteria can produce capsule, according to its capsule antigen It can be divided into 33 serotypes (1-31,33,1 / 2 types). According to the differences in the sequences of the seven housekeeping genes (aroA, cpn60, dpr, gki, mutS, recA, thrA), they can be divided into different genotypes (ST types). At present, at least 704 different genotypes have been found in the world Streptococcus suis of ST type, many ST types require cross-protection of Streptococcus suis vaccine. [0003] In 1954, Streptococcus suis was first reported as a pathogen, and its natural h...

Claims

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Application Information

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IPC IPC(8): C12N1/21G01N33/569A61K39/09A61P31/04C12R1/46
CPCA61K39/092A61K2039/522A61K2039/542A61K2039/552C07K14/315G01N33/56944
Inventor 李锦铨周洋王小红李志伟林蠡董星星
Owner HUAZHONG AGRI UNIV
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