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Swine erysipelas Spa A protein and application thereof to preparation of vaccines

A technology of Erysipelas suis and protein, applied in application, vaccines, veterinary vaccines, etc., can solve the problems of strong virulence of attenuated strains, weakened antigenicity, interference of attenuated vaccine efficacy, etc., and achieves convenient and accurate detection methods and antigen stability High and specific effect

Active Publication Date: 2019-08-30
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the inactivation process can cause the loss of bacterial antigenic epitopes or the weakening of antigenicity, inactivated vaccines cannot produce sufficient protection; the efficacy of attenuated vaccines is easily interfered by maternal antibodies and antibiotics, and attenuated strains have strong virulence and Possibility of Inducing Latent Infection
Moreover, the recessive infection of swine erysipelas may develop into a chronic type, and the existing vaccines cannot produce sufficient protection against chronic swine erysipelas

Method used

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  • Swine erysipelas Spa A protein and application thereof to preparation of vaccines
  • Swine erysipelas Spa A protein and application thereof to preparation of vaccines
  • Swine erysipelas Spa A protein and application thereof to preparation of vaccines

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Effect test

Embodiment 1

[0014] Embodiment 1: Amplification and sequence analysis of SpaA gene

[0015] In 2019, swine erysipelas was suspected to have occurred in a large-scale pig farm in Hubei Province, and many fattening pigs suffered acute illness and death. Redness is evident in the neck and ears, and purple plaques appear in the extremities and under the abdomen. After clinical investigation and laboratory testing, the preliminary diagnosis was swine erysipelas. After investigation, it is believed that the above-mentioned pig farms have been immunized with swine erysipelas inactivated vaccine and live attenuated vaccine before, and the detection of neutralizing antibodies confirmed that the immune effect has reached the standard. Therefore, it was suspected that Erysipelas suis had mutated under the selection pressure of the vaccine, and the lungs of dead pigs were aseptically collected for routine isolation and identification, and the pathogenic bacteria were determined to be Erysipelas suis....

Embodiment 2

[0026] Embodiment 2: Recombinant expression of Erysipelas suis SpaA protein

[0027] The preparation method of the SpaA protein comprises the following steps: a. constructing an expression vector; b. constructing an expression strain; c. inducing, extracting and purifying the recombinant SpaA protein.

[0028] a. Construct expression vector:

[0029] The positive clone plasmid pMD18-T-SpaA and the expression vector pET28a vector were double-digested with EcoRI and BamHI respectively, electrophoresed on 1.2% agarose gel, and then recovered with a DNA gel recovery kit to obtain about 5400bp and 1900bp fragments respectively. The pET28a expression vector was constructed by directional ligation at 16°C; after the sequence and reading frame were verified by sequencing, the plasmid was linearized and transformed into BL21 competent cells.

[0030] b. Construction of expression strains, protein extraction and purification:

[0031] After transformation, culture at 37°C for 18h. Pi...

Embodiment 3

[0033] Embodiment 3: the preparation of subunit vaccine

[0034] 1. Subunit vaccine preparation

[0035] 1. Preparation of SpaA protein for seedling production: inoculate BL21-SpaA strain in LB liquid medium containing kanamycin, shake and culture at 30°C for 18 hours, quantitatively aliquot, and use as first-class seeds after pure inspection. The first-grade seeds were inoculated in LB liquid medium, cultured with shaking at 37°C for 18 hours, and stored at 2-8°C after microscopic examination. Add LB liquid medium according to 60% (V / V) of the volume of the fermenter, and at the same time add antifoaming agent according to 0.1% (V / V) of the medium, pass high-temperature steam to sterilize for 30 minutes, and wait until the temperature of the medium drops to 37°C , inoculated with secondary seed liquid for SPAA protein production, the parameters of the fermenter were set to stir speed 800r / min, temperature 37°C, and maintain DO value (dissolved oxygen) at 20%. After 4 hours ...

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Abstract

The invention provides swine erysipelas Spa A protein. The amino acid sequence of the swine erysipelas Spa A protein is SEQ ID NO: 1. The erysipelothrix rhusiopathiae Spa A protein is used for preparing vaccines or diagnostic reagents. A subunit vaccine prepared from the erysipelothrix rhusiopathiae Spa A protein has the characteristics that antigen stability is high, purity is high, specificity is high, other uncorrelated antibodies are not generated, and the detection method is convenient and accurate, and a firm basis is established for industrial production of swine erysipelas subunit vaccines and diagnostic reagents.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a porcine erysipelas SpaA protein and its application in preparing vaccines. Background technique [0002] Swine erysipelas is a zoonotic infectious disease caused by Erysipelothrix rhusiopathiae, and its clinical features are mainly high fever, acute sepsis, skin rash, chronic verrucous endocarditis and skin necrosis with multiple nonseptic arthritis. At present, the disease is distributed in most countries in the world and occurs sporadically or epidemically, which has caused great economic losses to the pig industry and also threatened human health. Swine erysipelas, swine fever, and swine lung disease are known as the three major infectious diseases in China's pig industry, causing serious economic losses. [0003] At present, immunization is still the most effective way to prevent swine erysipelas, mainly using traditional vaccines, namel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31A61K39/02A61P31/04
CPCC07K14/195A61K39/0241A61P31/04A61K2039/552
Inventor 郭伟伟向银辉刘大卫邹桂荣刘新文蔡联燊范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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