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Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof

A technology for Streptococcus equi and attenuated vaccines, applied in biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve problems that hinder the development of attenuated vaccines, and achieve the effects of short cycle, convenient use, and low toxicity

Inactive Publication Date: 2012-10-10
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are too few studies on the virulence factors of this pathogen, which hinders the development of attenuated vaccines. In the prior art, only the M-like protein (SzP) has been studied more

Method used

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  • Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof
  • Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof
  • Capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Mutant strain C55138? has B build

[0033] 1. Materials: Streptococcus equi subsp. zooepidemicus C55138, purchased from China Veterinary Drug Control Institute. Plasmid pG + host5 was purchased from Appligene (Illkirch, France).

[0034] 2. Design and synthesis of primers

[0035] The genome structure of Streptococcus equi subsp. zooepidemicus C55138 (referred to as strain C55138) is attached figure 1 As shown, using the genomic DNA of strain C55138 as a template, use Primer Premier5.0 to design and amplify the target fragment has B Gene primers, where the hasBL primer pair contains restriction enzymes Sal I and Bam HI restriction site, the hasBR primer pair contains Bam HI and Eco Restriction site for RI. Primers were synthesized by Shanghai Sangong.

[0036] hasBL1: 5'-ATTTCTGTCGACGGCTCAGGATA-3' (SEQ ID NO: 1);

[0037] hasBL2: 5'-AATGGATCCTGACGCATTTAGGT-3' (SEQ ID NO: 2);

[0038] hasBR1: 5'-AACCATTACAATAACGGATCCTTTG-3' (SEQ ID NO: 3);

...

Embodiment 2

[0069] Embodiment 2 mouse virulence experiment

[0070] Twenty BALB / c mice aged 4-6 weeks were randomly divided into two groups, one group was injected with wild bacteria C55138, and the other group was injected with mutant bacteria C55138? has B , used to compare the virulence of the two strains.

[0071] After the bacteria were resuspended in PBS, the bacterial solution was properly diluted to 1×10 5 CFU / mL, mice were injected intraperitoneally with 500 μL, the survival status of the mice was observed, the death time of the mice was recorded, and the results were analyzed. The mice were continuously observed for 12 days.

[0072] See the experimental results Figure 5 : The mice in the wild bacteria group had a mortality rate of 80% within 9 days and showed severe clinical symptoms. The mutant bacteria C55138 ?hasB One group of mice survived 100% within 12 days without any clinical symptoms.

[0073] The experimental results showed that: mutant bacteria ?hasB virule...

Embodiment 3

[0074] Embodiment 3 mutant bacteria ?hasB Active immune protection experiment

[0075] Forty female BALB / c mice aged 4-6 weeks were randomly divided into 4 groups with 10 mice in each group. Group 1 mice were injected intraperitoneally with 500 μL mutant strain C55138 for the first time ?hasB For immunization, the bacterial concentration was 2×10 6 CFU / mL, immunized again with the same dose after 14 days; mice in the second group were used as positive controls to inject formaldehyde inactivated vaccine in the same way for immunization, the inactivated vaccine was emulsified with inactivated wild bacteria and Freund's adjuvant Inactivated vaccine, the first intraperitoneal injection of 500 μL, followed by immunization every 2 weeks. The mice in group 3 were injected with PBS emulsified with the same Freund's adjuvant in the same way as a negative control, and the mice in group 4 were injected with PBS in the same way as a blank control.

[0076] After all the mice in the ...

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Abstract

The invention discloses a capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain and a preparation method thereof, belonging to the technical field of animal vaccine preparation. The capsule deficiency type Streptococcus equi subsp. zooepidemicus attenuated vaccine strain is named Streptococcus equi subsp. zooepidemicus C55138 delta hasB, and was preserved in CCTCC (China Center for Type Culture Collection) on May 14, 2012 with the preservation number of CCTCC NO:M 2012164. The strain is constructed by hasB gene inactivation caused by allele replacing, is attenuated in toxicity, and can be used for preparing vaccine for Streptococcus equi disease. The vaccine production process is simple, the vaccine strain can be cultured on a large scale, the cost is low and the period is short; the vaccine presents cellular immunity and humoral immunity; and the vaccine is low in dosage, high in safety, low in toxicity and convenient for use, and can be popularized and used all over the world.

Description

technical field [0001] The invention relates to the technical field of animal vaccine preparation, in particular to an attenuated vaccine of capsular-deleted strain Streptococcus equi subspecies zooepidemicus and a preparation method thereof. Background technique [0002] Streptococcus equi subspecies zooepidemicus is a symbiotic bacterium that is symbiotic with the mucous membranes and skin of animals, especially in horses, and may cause sepsis, endocarditis and arthritis in many other animals, such as cattle, pigs, sheep, dogs . In China, Streptococcus equi subspecies zooepidemicus is an important pathogen that causes Streptococcus suis disease. In 1975, Streptococcus suis epidemic disease broke out in Sichuan, resulting in the death of 300,000 pigs, resulting in huge economic losses. So far, my country's pig industry is still plagued by this disease. [0003] Streptococcus equi subsp. zooepidemicus poses a serious threat to the whole pig industry in the world, but there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/09A61P31/04C12R1/46
Inventor 陈瑶生魏子贡付强刘小红莫德林
Owner SUN YAT SEN UNIV
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