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Streptococcus suis B cell dominant epitope tandem vaccine and preparation method thereof

A dominant epitope, Streptococcus suis technology, applied in chemical instruments and methods, antibacterial drugs, pharmaceutical formulations, etc., can solve problems such as unanalyzed design, and achieve convenient and safe preparation process, good stability, and high safety Effect

Active Publication Date: 2019-01-11
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Prior art shows that phosphate-3-glyceraldehyde dehydrogenase (glyceraldehyde-3-phosphatedehydrogenase, GAPDH), lysozyme-released protein (Muramidase-released protein, MRP) and dihydrolipoamide dehydrogenase (Dihydrolipoamide dehydrogenase, DLDH) They are three important protective antigens in Streptococcus suis. However, there is no method to analyze and design B cell dominant antigenic epitopes for the above three antigen molecules, especially the combination of the three antigen molecules, and to produce genetically engineered vaccines. Related reports

Method used

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  • Streptococcus suis B cell dominant epitope tandem vaccine and preparation method thereof
  • Streptococcus suis B cell dominant epitope tandem vaccine and preparation method thereof
  • Streptococcus suis B cell dominant epitope tandem vaccine and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0056] Epitope Screening

[0057] (1) Protein primary structure analysis

[0058] According to the GAPDH, MRP, and DLDH protein amino acid sequences of Streptococcus suis ZY05719 strain published by NCBI (accession numbers are AKG39592.1, AKG40097.1, and AKG41058.1), the online TMHMM tool (http: / / www.cbs.dtu .dk / services / TMHMM / ) to analyze the transmembrane region of the protein, and use the online SignalP tool http: / / www.cbs.dtu.dk / services / SignalP / to analyze the signal peptide region of the protein.

[0059] The results predicted by TMHMM tool analysis are: GAPDH protein has no transmembrane region, and its 1-336 amino acid sequences are all extracellular fragments (such as figure 1 shown); MRP protein has a transmembrane region located at amino acids 23-45, such as figure 2 As shown, the N-terminus of the protein is inside the cell, and the 46-1256 amino acid is outside the cell; the 1-586 amino acid of the DLDH protein is an extracellular fragment, and there is no tran...

Embodiment 2

[0069] Tandem epitope vaccine design and plasmid construction

[0070] According to the predominant B cell antigen epitopes of GAPDH, MRP, and DLDH proteins predicted in Example 1, all epitopes were combined and spliced ​​in sequence according to the order of GAPDH-MRP-DLDH, and GGGG flexible fragments were used as linker amino acids between polypeptides to reduce Interactions between epitopes. The amino acid sequence of the GAPDH-MRP-DLDH tandem epitope protein is shown in SEQ ID NO: 2, and the amino acid number of the recombinant protein is 291aa. According to the original reference sequence of GAPDH (Genbank accession number: ZY05719_00895), MRP (Genbank accession number: ZY05719_03650), DLDH (Genbank accession number: ZY05719_08710), the above tandem sequences were codon optimized and BamHI and XhoI were introduced at the N-terminus and C-terminus respectively Restriction sites, the nucleotide sequence of the GAPDH-MRP-DLDH tandem epitope protein finally obtained is 888bp...

Embodiment 3

[0072] Prokaryotic expression and purification of tandem epitope protein GMD

[0073] (1) Prokaryotic expression of epitope protein. The recombinant plasmid pET-28a(+)-GMD constructed in Example 2 was heat-shocked at 42°C to transform E. coli competent cells BL21 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and the recovered competent bacteria were subjected to 5000 rpm After centrifugation for 5 minutes per minute, the cells were resuspended in 100 μL of LB liquid medium, and the resuspended cells were spread on LB plates with kanamycin and cultured overnight in a 37°C incubator. Single clones on the plate were selected with a sterile pipette tip, placed in 1 mL of LB liquid medium containing kanamycin (50 μg / mL), and cultured on a constant temperature shaker at 37°C at 220 rpm for 4 hours. Colony PCR method was used for cloning identification. The primers used in PCR were: upstream primer GMD-F: 5'-ACGGGATCCGAACCGGGTAATATT-3', downstream primer GMD-R: 5'-ACG...

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Abstract

The invention discloses a streptococcus suis B cell dominant epitope tandem protein, relates to a method for preparing a streptococcus suis B cell dominant epitope tandem vaccine and discloses a use of a B cell dominant epitope tandem gene and a fusion protein in preparation of a vaccine. The method for preparing the vaccine comprises selecting B cell dominant epitopes of streptococcus suis protective antigens GAPDH, MRP and DLDH through software, connecting amino acid sequences as GGGG flexible fragments in series, carrying out prokaryotic expression through the recombinant expression vectorformed from the fragments in series, purifying the expressed fusion protein, and processing the fusion protein and a Freund's adjuvant into a vaccine preparation. The mouse immunogenicity evaluation result shows that the vaccine can effectively protect the streptococcus suis type 2. Compared with the traditional inactivated vaccine and attenuated vaccine, the genetic engineering vaccine provided by the invention has the advantages of convenient and safe preparation process, high production efficiency, high product purity, good stability, high yield and high safety.

Description

technical field [0001] The invention relates to the field of genetic engineering subunit vaccine production, in particular to a Streptococcus suis B cell dominant epitope tandem vaccine and a preparation method thereof. Background technique [0002] Streptococcus suis is an important zoonotic pathogen, which can cause diseases such as meningitis, arthritis, endocarditis and sepsis in pigs. At present, 35 serotypes (1-34, 1 / 2) are known, among which types 1, 2, 7, and 9 are pathogenic types, and type 2 is the most pathogenic, and it is the most popular in my country. Group carrier rate is high (XU J, MU Y, ZHANG Y, et al. Antibacterial effect of porcine PTX3 against Streptococcus suis type2 infection. [J] Microb Pathog, 2015, 89: 128-139). Streptococcus suis outbreaks broke out in Jiangsu in 1998 and Sichuan in 2005, which had a great impact on my country's pig industry (YU H, JING H, Chen Z, et al. Human Streptococcus suis outbreak, Sichuan, China. [J] Emerg Infect Dis, 2006...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70A61K39/09A61P31/04
CPCA61K39/092A61K2039/523A61P31/04C07K14/315C07K2319/40C12N15/62C12N15/70
Inventor 孙建和孔里程严亚贤王兆飞杨登辉
Owner SHANGHAI JIAO TONG UNIV
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