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Erysipelothrix rhusiopathiae subunit vaccine, preparation method and application

A technology of Erysipelas erysipelas and subunit vaccine, which is applied in the field of vaccine preparation of livestock infectious diseases, can solve the problems of unclear mechanism, weakened virulence, and interfere with the efficacy of vaccines, so as to achieve the effect of safety and avoid invasion.

Active Publication Date: 2016-11-23
HUAZHONG AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Maternal antibodies and drugs can easily interfere with the efficacy of the vaccine, and if the live attenuated strain returns to the virus, it poses a great risk to susceptible pigs
Although many attenuated live vaccines have been cultured in rabbits, chicken embryos, air-dried or in media containing acridine dyes, the mechanism of attenuation of virulence is unclear

Method used

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  • Erysipelothrix rhusiopathiae subunit vaccine, preparation method and application
  • Erysipelothrix rhusiopathiae subunit vaccine, preparation method and application
  • Erysipelothrix rhusiopathiae subunit vaccine, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Obtaining the protein sequence of spaA specific gene

[0036] Our laboratory isolated a JZ08 strain from clinically dead pigs, which was identified as Erysipelothrix rhusiopathiae by PCR. The primers in Table 1 were used to amplify, and the nucleotide sequence corresponding to the truncated protein spaA of the present invention was obtained as shown in SEQ ID NO.1.

[0037] Download the full gene sequence of spaA published by NCBI and construct the phylogenetic tree. The phylogenetic tree clearly shows that the spaA sequence used in this study is different from the sequence previously published on NCBI. figure 1 .

[0038] 2) The sequence of the truncated protein spaA

[0039]The sequence of the truncated protein spaA of the present invention is shown in SEQ ID NO.2.

[0040] Build a phylogenetic tree

[0041] Phylogenetic tree analysis with the spaA protein sequence known on the Internet, it is found that the protein sequence used in this study is located on the sa...

Embodiment 2

[0043] Construction of Escherichia coli BL21 / pET28a-spaA:

[0044] The plasmid pET-28a (+) that the present invention adopts is purchased from Noagen company (this pET-28a (+) plasmid map is as follows figure 2 Shown) Competent Escherichia coli BL21 (DE3) was purchased from Wuhan Life Technology Co., Ltd., Hubei Province, China.

[0045] Primer design and synthesis

[0046] Primer design software Primer 5.0 was used to design primers for amplifying spaA (Table 1). The primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd.

[0047] Table 1: Primer design for cloning the antigenic protein gene of the present invention

[0048]

[0049] The genome of the extracted JZ08 strain was used as a PCR template. PCR amplification was performed using the primers shown in Table 1. The PCR reaction system is as follows:

[0050]

[0051]

[0052] The reaction program was: pre-denaturation at 94°C for 5 min; 94°C for 1 min; 55°C for 30 s; 72°C for 1 min...

Embodiment 3

[0064] Preparation method of recombinant protein:

[0065] 1) Escherichia coli (Escherichia coli) BL21 / pET28a-spaA was inoculated in 3 mL LB liquid medium containing 25 μg / ml Kan, 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 25 μg / ml Kan, shake and culture at 37°C for about 3 hours, until OD 600 When it reaches 0.8, add IPTG to the final concentration of 0.8mmol / L, continue to cultivate for 3h, and then collect the bacteria.

[0066] 2) SDS-PAGE electrophoresis analysis of expression products

[0067] The induced recombinant Escherichia coli was centrifuged at 8000r / min for 15min. The pellet was resuspended with 1 / 10 volume of 50mM Tris-cl (pH 8.0), and lysozyme was added to a final concentration of 1mg / ml, and kept on ice for 30min. Ultrasonic crushing was carried out under ice bath conditions until the bacterial liquid was no longer viscous, centrifuged at 10000r / min for 30m...

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Abstract

The invention provides an erysipelothrix rhusiopathiae subunit vaccine, a preparation method and application. The subunit vaccine contains an amino acid sequence shown as SEQ ID NO.2, the sequence is different from any disclosed related sequences, a nucleotide sequence corresponding to protein is cloned to escherichia coli, and then a genetically engineered bacterium-escherichia coli BL21 / pET-28a-spaA capable of expressing spaA protein is obtained, wherein the preservation number is CCTCC NO:M2015076. The genetically engineered bacterium is high in protein expression quantity, easy to operate and suitable for large-scale subunit vaccine production. The prepared vaccine is high in antibody level generated to an organism and long in continuity, has the safety and the stability and is the erysipelothrix rhusiopathiae subunit vaccine which extremely has development potential.

Description

technical field [0001] The invention relates to the technical field of preparation of livestock infectious disease vaccines. It specifically relates to a subunit vaccine of Erysipelothrix rhusiopathiae and its preparation method and application. The erysipelothrix subunit vaccine provided by the invention can improve the ability of pigs to resist erysipelothrix. Background technique [0002] Porcine erysipelas mainly causes septicemia, endocarditis and polyarthritis in pigs. Most of them occur in pigs and pregnant sows aged 3-6 months. Adult pigs and old pigs are less common. Its prevalence has a certain season. It is most popular in hot and rainy seasons, and it is mostly sporadic or endemic, and it can occasionally break out. There are 4 species of Erysipelothrix, one of which is Erysipelothrix rhusiopathiae, which has serotypes 1a, 1b, 2, 4, 5, 6, 8, 11, 12, 15, 16, 17, 19, 21 and N; the second is Erysipelothrix tonsillarum with serotypes 3, 7, 10, 14, 20, and 23; the ...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N1/21A61K39/02A61K48/00A61P31/04C12R1/01
Inventor 金梅林李敬涛王雅康超孙小美朱伟峰吴超
Owner HUAZHONG AGRI UNIV
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