Paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof

A Streptococcus iniae, multi-epitope technology, applied in the direction of fusion of polypeptides, peptides, bacteria, etc., can solve the problems of epitope vaccine reports and unsatisfactory effects, and achieve strong immune protection

Active Publication Date: 2019-10-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Studies on antigenic proteins of Streptococcus iniae have shown that although MtsB, Sip11, FBA, CAMP, SiM protein, C5a peptidase, interleukin-8 protease, enolase, capsular polysaccharide and PGM can induce host immune response, but Not as effective as a vaccine
Current studies on S. iniae vaccines include formalin inactivated vaccines, modified live vaccines, DNA vaccines, and subunit vaccines, but there is no report on epitope vaccines

Method used

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  • Paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof
  • Paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof
  • Paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Design of molecular structure of Streptococcus iniae multi-epitope polypeptide

[0032] (1) Taking the amino acid sequence of Streptococcus iniae GAPDH protein (ACX85247.1) as the analysis material, the secondary structure of Streptococcus iniae GAPDH protein was predicted by SOPMAServer. The results showed that in the GAPDH gene, random coils accounted for 36.62%, β-turns accounted for 7.32%, α-helixes and extended chains (beta-sheets) accounted for 30.57% and 25.48% respectively. For distribution, see figure 1 .

[0033] (2) Apply DNAStar Protean bioinformatics software and online website (IEDB: http: / / tools.iedb.org / main / bcell / and ProtScale: http: / / us.expasy.org / ) to analyze protein secondary structure The flexible region and plasticity region of the predicted amino acid hydrophilicity (Hydrophilicity Plot-Kyte-Doolittle), flexibility (Flexible Regions-Karplus-Schulz), antigenicity (Antigenic Index-Jameson-Wolf) and surface accessibility (Surface Pr...

Embodiment 2

[0043] Example 2: Construction and induced expression of recombinant expression vector pET-28a-MEPIG

[0044] 1. Construction of recombinant expression vector

[0045] The Streptococcus iniae multi-epitope vaccine molecule designed above is named MEPIG, its amino acid sequence is SEQ ID NO: 8, and the nucleotide sequence corresponding to the linker polypeptide is reversed to the corresponding gene according to the nucleotide tropism of the E. coli expression system Sequence (SEQ ID NO: 9), and BamHI / SacI and SacI / HindIII restriction sites, protective bases and stop codons were added to the 5' and 3' ends of the tandem multi-epitope MEPIG gene sequence respectively. After codon optimization, it was sent to Shanghai Sangon Bioengineering Co., Ltd. for synthesis and cloned into the pET-28a plasmid to form a recombinant plasmid.

[0046] 2. Induced expression of recombinant multi-epitope polypeptides

[0047] 1) Inoculate the positive bacteria containing the recombinant plasmid ...

Embodiment 3

[0049] Example 3: Purification and immunogenicity detection of recombinant multi-epitope polypeptides

[0050] (1) Purification of recombinant protein

[0051] Centrifuge the bacterial solution confirmed to be successfully induced with a centrifuge (8000×g, 10min, 4°C), discard the supernatant, collect the bacterial cells, wash once with PBS, and use about 40-50mL of Binding buffer (Na 2 HPO 4 14.5g; NaH 2 PO 4 1.48g; NaCl 29.3g; urea 480g; 40mM imidazole; pH 7.4, dilute to 1L with ultrapure water, and filter with membrane) to resuspend, place on ice, and crush with ultrasonic breaker (3s on, 3s off, power 37% ) to light yellow clear liquid, after centrifugation at 8000×g for 5min, take the supernatant, and store it at 4°C for later use; connect the sample cup, His-tagged protein affinity chromatography column, etc., and check the airtightness of the device; wash with ultrapure water Instrument pipeline A, B and protein affinity chromatography column, and then use the pr...

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Abstract

The invention provides a paralichthys olivaceus streptococcus iniae GAPDH series-connection multi-epitope polypeptide and application thereof. The provided series-connection multi-epitope polypeptideis formed by binding four antigen epitope peptides through peptide joints, wherein the amino acid sequences of the four antigen epitope peptides are shown in SEQID NO:1-4 respectively. One specific amino acid sequence of the series-connection multi-epitope polypeptide is SEQID NO:8. The invention further provides a streptococcus iniae GAPDH multi-epitope vaccine prepared by using the series-connection multi-epitope polypeptide as an antigen and a vaccine adjuvant. The multi-epitope vaccine can provide strong immune protection for paralichthys olivaceus resistance to S.iniae infection, and theeffect is significantly higher than that of a GAPDH recombinant protein subunit vaccine and a S.iniae inactivated vaccine.

Description

technical field [0001] The invention belongs to the technical field of aquatic vaccines, and in particular relates to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) tandem multi-epitope polypeptide of Streptococcus iniae and its application in preparing vaccines. Background technique [0002] Streptococcus iniae (S. iniae) is a Gram-positive bacterium belonging to the genus Streptococcus of the family Streptococcus. Streptococcus iniae is widely distributed in marine and freshwater environments, and can infect a variety of marine and freshwater farmed fish, such as flounder (P.olivaceus), turbot (Scophthalmus maximus), red sea bream (Chrysophrys major), black sea bream (Sparus microcephalus) , Tilapia (Oreochromis nilotica), etc., can be infected from juvenile to adult fish, causing significant economic losses to the aquaculture industry. S. iniae can also infect humans, causing bacterial cellulitis, meningitis, endocarditis, and infectious arthritis. At present, antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/11C12N15/70C12N1/21A61K39/09A61P31/04C12R1/19
CPCC07K7/08C12N15/70A61K39/092A61P31/04C07K2319/00A61K2039/70
Inventor 绳秀珍刘敏战文斌唐小千邢婧
Owner OCEAN UNIV OF CHINA
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