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Gene gapdh for expressing erysipelothrix rhusiopathiae recombinant protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as well as recombinant escherichia coli and applications of gene gapdh

A technology for recombining Escherichia coli and Erysipelas suis, applied in the direction of recombinant DNA technology, application, genetic engineering, etc., can solve the problems of limited protective antigens and affecting the creation of new subunit vaccines for erysipelas suis

Inactive Publication Date: 2018-05-01
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Currently known protective antigens are very limited, which seriously affects the creation of new subunit vaccines against porcine erysipelas

Method used

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  • Gene gapdh for expressing erysipelothrix rhusiopathiae recombinant protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as well as recombinant escherichia coli and applications of gene gapdh
  • Gene gapdh for expressing erysipelothrix rhusiopathiae recombinant protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as well as recombinant escherichia coli and applications of gene gapdh
  • Gene gapdh for expressing erysipelothrix rhusiopathiae recombinant protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as well as recombinant escherichia coli and applications of gene gapdh

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of recombinant Escherichia coli expressing Erysipelas suis recombinant protein GAPDH

[0041] 1 Materials and methods

[0042] 1.1 Strains and growth conditions

[0043] Bacterial strain adopts local virulent endemic strain SE38 of Erysipelothrida suis (hereinafter referred to as SE38 strain, see literature "analysis of drug resistance of Erysipelothrix rhusiopathiae isolates in some areas of Hubei"); TSB medium or TSA medium (BD Company); the carrier is Escherichia coli pET-28a, and the competent cells are Escherichia coli BL21(D3), all purchased from TransGene Company.

[0044] 1.2 Reagents

[0045] Genome extraction kit was purchased from TIANGEN company

[0046] The following primer pair was designed using the gene gapdhSEQ ID NO.1 as a template, and the primer pair was synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0047] Forward primer C1 (SEQ ID NO.3) is shown as SEQ ID NO.3 in the sequence listing:

[00...

Embodiment 2

[0061] Preparation of embodiment 2 recombinant protein

[0062] Utilize above-mentioned recombinant Escherichia coli BL21(DE3)-gapdh to prepare recombinant protein: specific method:

[0063] 1) Recombinant Escherichia coli BL21(DE3)-gapdh was streaked on the LB plate containing kanamycin resistance, cultured at 37°C for 12-14h, and a single colony was picked and cultured in 5ml LB liquid containing kanamycin resistance medium, 37°C, 220r / min shaking culture for 12-14h, inoculate the bacterial solution into 1L LB liquid medium containing kanamycin resistance at a ratio of 1:200, and culture at 37°C until OD 600 When it reaches 0.8, add a final concentration of 200mmol / L IPTG to select a line for induction, continue to cultivate overnight, and collect the bacteria by centrifugation at 5000r / min at room temperature for 15min; at the same time, do a non-induction control test.

[0064] 2) Add 40ml of Ni-NTA binding buffer to each 1L of bacteria, mix well, and sonicate in an ice b...

Embodiment 3

[0066] Example 3 SDS-PAGE and immunoblotting analysis

[0067] 1. SDS-PAGE and western blot analysis

[0068] The purified recombinant protein GAPDH and the unpurified crude protein were electrophoresed on a 12% PAGE gel after SDS treatment. After the electrophoresis, the recombinant protein GAPDH was transferred to a nitrocellulose membrane by a semi-dry method, and then His monoclonal antibody was used to Or swine erysipelas recovered pig serum was used as the primary antibody, and goat anti-mouse or goat anti-pig IgG(H+L)-HRP was used as the secondary antibody for western blot analysis.

[0069] 2. Result judgment

[0070] 2.1 Perform PCR amplification to obtain a base sequence of 1023bp, which contains the full-length ORF (SEQ ID NO.1) of E. suis GAPDH, and combine the deduced Erysipelotus suis GAPDH amino acid sequence (SEQ ID NO.1) with the GAPDH of Streptococcus pyogenes The amino acid sequence (Genbank sequence number AAK33348) was compared and found that there was a...

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Abstract

The invention discloses a gene gapdh for expressing erysipelothrix rhusiopathiae recombinant protein GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as well as recombinant escherichia coli and applications of the gene gapdh. According to the technical scheme, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of erysipelothrix rhusiopathiae is cloned, expressed and purified. The western blot analysis for the porcine erysipelas rehabilitation pig serum shows that the recombinant protein GAPDH has very good immunogenicity. The mouse experiment shows that the recombinant protein GAPDH caninduce generation of high-titer antibodies and generate effective protection force. A novel candidate antigen is provided for the creation of a novel porcine erysipelas vaccine.

Description

technical field [0001] The invention relates to the field of vaccine genetic engineering, in particular to a gene gapdh expressing Erysipelas suis recombinant protein GAPDH, its recombinant Escherichia coli and its application. Background technique [0002] Animal erysipelasia is a bacterial infectious disease caused by Erysipelothrix rhusiopathiae in various animals (including humans). Swine erysipelas is mainly manifested as septicemia, endocarditis and polyarthritis in pigs, which brings huge economic losses to the pig industry. Porcine erysipelas used to be in a sporadic state, but since 2010, the incidence of porcine erysipelas has shown an increasing trend year by year, and it is mainly caused by large-scale pig farms, resulting in huge losses. [0003] Due to the abuse of antibiotics, drug-resistant strains of Erysipelas suis are produced, making drug treatment tend to be ineffective; antibiotic drug residues pose a major threat to the food safety of pork in our coun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12N15/66C12N1/21A61K39/02A61P31/04C12R1/19
CPCC12N9/0008A61K39/0241A61K2039/552C12N15/66C12N15/70C12Y102/01
Inventor 金梅林吴超朱伟峰李敬涛康超蔡承志孙小美
Owner HUAZHONG AGRI UNIV
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