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Streptococcus-suis-disease-resistant fusion protein with autoimmune activity and preparing and application thereof

A fusion protein, Streptococcus suis technology, applied in the field of fusion proteins, can solve the problems of lack of immune adjuvant, no cross protection, no reliable preventive effect, etc.

Active Publication Date: 2016-11-16
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The inactivated vaccines or live attenuated vaccines currently used in pig farms have no reliable preventive effect, and most of them have no cross-protection effect between different serotypes, and their safety is poor
[0007] Although a large number of potential subunit vaccines have emerged in recent years, there is a lack of ideal and suitable immune adjuvants. Therefore, a subunit vaccine with high safety, cross-protection against multiple subtypes of Streptococcus suis, and autoimmune adjuvant effects has been developed , has a broad market prospect

Method used

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  • Streptococcus-suis-disease-resistant fusion protein with autoimmune activity and preparing and application thereof
  • Streptococcus-suis-disease-resistant fusion protein with autoimmune activity and preparing and application thereof
  • Streptococcus-suis-disease-resistant fusion protein with autoimmune activity and preparing and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] [Example 1] Sao 28‐318 and Omp16 26‐168 / Sao 28‐318 Construction of recombinant protein

[0065] 1. Design and synthesis of primers

[0066] According to published sao on GenBank 28‐318 (GenBank Gene ID: 61969363), omp16 26‐168 (GenBank Gene ID: 333601408) gene sequence, after splicing, was synthesized by Nanjing GenScript Biotechnology Co., Ltd. to obtain the following sequence Omp16 26‐168 / Sao 28‐318 , design and synthesize two pairs of primers, the primers were synthesized by Shanghai Sangon Bioengineering Technology Co., Ltd., the sequences of the primers are as follows:

[0067] 1) sao 28‐318 Gene PCR Amplification Primers

[0068] Upstream primer P1

[0069] 5'-CGCCCATGGCGTCGGCACAAGAAGTAA-3'

[0070] downstream primer P2

[0071] 5'-CGCCTCGAGCTCTTCATTAGCTATTTGC-3'

[0072] 2) Omp16 26‐168 / Sao 28‐318 Gene PCR Amplification Primers

[0073] Upstream primer P1

[0074] 5'-CGCCCATGGCGGCGTCAAAAGAAGAACCT-3'

[0075] downstream primer P2

[0076] 5'-C...

Embodiment 2

[0103] [Example 2] Identification of antigenicity of fusion protein

[0104] The fusion protein prepared above was subjected to SDS-PAGE and the gel was transferred to PVDF membrane by semi-dry method, blocked at 37°C for 1.5h, and primary antibody (the mouse serum collected after the second immunization was diluted by a certain factor) was added at 4 ℃ blocked overnight. After the membrane was washed with TBST, the secondary antibody goat anti-mouse (IgG‐HRP) diluted at 1:2000 was added, and placed on a shaker at room temperature for 2 hours. After the secondary antibody was washed away, the two primary antibodies and the fusion protein were detected with a chemiluminescent substrate. response situation. The results showed that corresponding bands appeared at the position of the size of the target protein, indicating that the fusion protein could specifically react with mouse serum and had good immunogenicity.

Embodiment 3

[0105] [Example 3] fusion protein Omp16 26‐168 / Sao 28‐318 Can induce a strong humoral immune response

[0106] Detection of antibody levels induced by fusion proteins

[0107] Detection of anti-Omp16 in serum of immunized mice by conventional ELISA 26‐168 / Sao 28‐318 Specific antibody levels. Specifically, to artificially synthesize and identify the correct Omp16 26‐168 / Sao 28‐31 8 Coat the 96-well ELISA plate, overnight at 4°C; wash the plate 3 times with PBST, add blocking solution and incubate at 37°C for 2 hours; wash 3 times with PBST, dilute the serum to be tested with PBST buffer 1:100, and incubate at 37°C 1.5h. After washing the plate 3 times, add HPR-labeled goat anti-mouse IgG-HRP and incubate at 37°C for 1 h; wash the plate 5 times with PBST, develop color with TMB in the dark, 2mol / L H 2 SO 4 The reaction was terminated, and the OD value at 450nm was read with a microplate reader to determine the serum antibody level. The results showed that the fusion...

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Abstract

The invention discloses a streptococcus-suis-disease-resistant fusion protein with the autoimmune activity and preparing and application thereof. The fusion protein is composed of Omp16<26-168> peptide located at the amino terminal and Sao<28-138> peptide located at the carboxyl terminal. The Sao<28-138> peptide is an amino acid fragment which is exposed out of a streptococcus suis cell in a Sao protein molecule, has the immunogenicity, and is highly conservative between different strains and subtypes, and the Omp16<26-168> peptide is an amino acid fragment is exposed out of a Brucella cell in a Omp16 molecule, has the TLR4 activating activity, and is free of Lipobox (the Lipobox is lowly toxic or completely nontoxic). Mice are subjected to an immunity virus attacking test through the Omp16<26-168> / Sao<28-138> fusion protein, it is shown that the Omp16<26-168> / Sao<28-138> fusion protein has the good immune protection effect, a quite-high cellular immune response and a quite-high humoral immune response can be induced, but a Th1 response is the main response, and the fusion protein has the self-adjuvant effect, is an ideal vaccine antigen of streptococcus, and has the important value in development and application aspects of novel vaccine.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a fusion protein resistant to streptococcus suis and having autoimmune activity, as well as its preparation and application. Background technique [0002] Streptococcus suis is one of the main pathogenic bacteria in pigs, and there are as many as 35 serotypes. Among the streptococci isolated from clinical pigs, type 2 is the most common and the most pathogenic serotype type. Clinically, streptococcal suis mainly manifests as meningitis, lymph node abscess, arthritis, suppurative pneumonia and sepsis. Under the action of some specific inducements, the mortality rate of affected pigs can reach 80%. [0003] In recent years, due to the increasing stocking density in commercial pig farms, suis streptococcal disease is on the rise, often secondary to some viral diseases or mixed with some bacterial diseases, not only causing serious economic losses to pig production, bu...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/09A61K39/39A61P31/04
CPCA61K39/092A61K39/39A61K2039/55516A61K2039/57C07K14/23C07K14/315C07K2319/00C12N15/70C12N2800/101
Inventor 陈小军孙志良李元义
Owner HUNAN AGRICULTURAL UNIV
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