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A multi-epitope polypeptide of flounder streptococcus pdha1

A Streptococcus iniae, multi-epitope technology, applied in the direction of antibacterial drugs, bacterial antigen components, antibody medical components, etc., to achieve a strong immune protection effect

Active Publication Date: 2022-04-12
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are research data on formalin inactivated vaccine, modified live vaccine, DNA vaccine and subunit vaccine of Streptococcus iniae, but there is no report on epitope vaccine of Streptococcus iniae

Method used

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  • A multi-epitope polypeptide of flounder streptococcus pdha1
  • A multi-epitope polypeptide of flounder streptococcus pdha1
  • A multi-epitope polypeptide of flounder streptococcus pdha1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Bioinformatics analysis of B cell linear epitopes of PDHA1 protein

[0023] 1. Predicted epitopes

[0024] 1) Using the amino acid sequence of Streptococcus iniae PDHA1 protein (WP_003099247.1) as the analysis material, the secondary structure of Streptococcus iniae PDHA1 protein was predicted using SOPMAServer. The results showed that random coils accounted for 25.78% and β-turns accounted for 13.04% of the PDHA1 gene. The α-helix and extended chain, namely β-sheet, account for 45.65% and 15.53% respectively. For the distribution of various structures in the amino acid sequence of Streptococcus iniae PDHA1 protein, see figure 1 .

[0025] 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 Flexible and plastic regions, predicting amino acid hydrophilicity (Hydrophilicity Plot-Kyte-Doolittle), flexibility (Flexible Regions-Ka...

Embodiment 2

[0045] Example 2: Design of tandem multi-epitope polypeptide molecular structure and construction of recombinant expression vector

[0046] 1. Construction of tandem multi-epitope polypeptides

[0047] (1) The identified antigenic epitopes are connected in series using different polypeptide linkers (GGGG, EAAAK, AAY, KK, GPGPG) and different connection methods to determine the best connection method. The results show that after PDHA1 is connected in series with the connection sequence of KK linker (P1-KK-P2-KK-P3-KK-P4), each epitope is relatively independent, and the antigenic parameters are good ( Figure 4 ). The tandem multi-epitope polypeptide is named MEPIP, and its amino acid sequence is SEQ ID NO:5:

[0048] NHRGHGQSIAKDMDKKAGKATGVSKGRGGSKKKYRTKEEVDAWKEKKKRAYLTAEGIATDEE.

[0049] (2) Obtain the gene sequence of each epitope, and at the same time reverse the nucleotide sequence corresponding to the linker polypeptide into the corresponding gene sequence according to ...

Embodiment 3

[0072] Example 3: Application of PDHA1 multi-epitope vaccine in providing immune protection for flounder against S.iniae infection

[0073] (1) Immunization and sampling of flounder

[0074] ①Experimental settings The recombinant protein (rMEPIP) of the multi-epitope polypeptide MEPIP prepared in Example 2, the recombinant subunit vaccine (rPDHA1), inactivated Streptococcus iniae (FKC) and the PBS negative control consisted of 4 groups, each group of flounder 100 Finally, the protein concentration was determined by BCA method and the protein concentration was adjusted to 2 mg / mL with sterile PBS, and the vaccine protein and Freund's complete adjuvant were lubricated with a syringe lubricated with Freund's complete adjuvant, respectively, connected with a connecting tube, and mixed with an injection emulsifier until the mixture does not diffuse in water. The negative control group was injected with an equal volume of adjuvant and PBS.

[0075] ② Before immunization and 1d, 3d...

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Abstract

The invention provides a multi-epitope polypeptide of flounder Streptococcus dolphin PDHA1, which comprises four Streptococcus iniae PDHA1 protein epitope peptides connected by polypeptide linkers, and the amino acid sequences of the Streptococcus iniae PDHA1 protein epitope peptides are respectively is SEQ ID NO: 1-4. Another aspect of the present invention provides a Streptococcus iniae PDHA1 multi-epitope vaccine, wherein the antigen is the above-mentioned multi-epitope polypeptide. The PDHA1 multi-epitope vaccine prepared by the invention can provide strong immune protection for flounder against S. iniae infection, and the protection effect is obviously better than PDHA1 recombinant subunit vaccine and S. iniae whole bacteria inactivated vaccine.

Description

technical field [0001] The invention belongs to the technical field of immune antigen preparation, in particular to a multi-epitope polypeptide of Pyruvate dehydrogenase E1α subunit (PDHA1) of Streptococcus iniae, and the use of the multi-epitope polypeptide in preparing vaccines application. Background technique [0002] Streptococcus dolphin (S. iniae) is a Gram-positive bacterium, oval in shape, with a diameter of about 1.5 μm, capsuled and arranged in chains, belonging to the genus Streptococcus of the family Streptococcus. Streptococcus iniae is widely distributed in sea and freshwater environments, and can infect a variety of sea and freshwater farmed fish such as flounder (Paralichthys olivaceus), turbot (Scophthalmus maximus), red sea bream (Chrysophrysmajor), tilapia (Oreochromis nilotica), etc. Aquaculture causes significant economic losses. S. iniae can also infect humans, causing bacterial cellulitis, meningitis, endocarditis, and infectious arthritis. The use...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53A61K39/09A61P31/04
CPCC12N9/0008A61K39/092A61P31/04C12Y102/01051
Inventor 绳秀珍刘敏战文斌唐小千邢婧
Owner OCEAN UNIV OF CHINA
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