Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator

A technology of ompas-19 and outer membrane protein, applied in the field of bacterial outer membrane protein ompAs-19 and its application as an immune regulator, can solve the problem of low immune regulation function and achieve high immune protection effect

Inactive Publication Date: 2016-05-11
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the outer membrane protein OmpA only has a good immune regulation function on the same bacteria, and has a low immune regulation function on other bacteria.
However, there is no report on the development of multi-effect and multivalent vaccines obtained by this technology

Method used

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  • Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator
  • Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator
  • Bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator

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Experimental program
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Effect test

Embodiment 1

[0023] Bacterial outer membrane protein OmpA gene shuffling template: 6 bacterial outer membrane protein OmpA genes were selected as shuffling objects, which were respectively derived from 4 kinds of bacteria including Vibrio alginolyticus, Vibrio parahaemolyticus, Edwardsiella tarda and Escherichia coli. The six OmpA genes are Vibrio alginolyticus 0764 (VA0764), 1186 (vpa1186), Vibrio parahaemolyticus 0764 (VP0764), 1186 (VPA1186), Edwardsiella tarda ompA (ETAE_1267), Escherichia coli ompA (E. coliompA), the gene length is between 960bp and 1056bp. Sequence alignment analysis of these 6 OmpA genes using DNAman software found that their homology was 65.59%, the results are shown in figure 1 , which can be used for gene shuffling research.

Embodiment 2

[0024] Obtaining of embodiment 2 bacterial outer membrane protein OmpA shuffling gene

[0025] PCR amplification of the shuffled template: primers were designed according to the 6 OmpA gene sequences published by NCBI, and the primer sequences are shown in Table 1. By analyzing the 6 OmpA gene sequences, there is a BamHI restriction site at 760bp of Edwardsiella tarda ompA (ETAE_1267) and 745bp of Escherichia coli ompA (E.coliompA). Because the prokaryotic expression vector used later will use this restriction site, the two genes use the BamHI restriction site as the dividing point, and design two pairs of primers respectively to amplify the E.coliompA and ETAE_1267 genes into two fragment.

[0026] Table 1 is used for the primer sequence of shuffling ompA template amplification

[0027]

[0028] E.coliompA-1 is from 1 to 745bp, E.coliompA-2 is from 746 to 1041bp, E.tardaompA-1 is from 1 to 760bp, and E.tardaompA-2 is from 761 to 1056bp.

[0029] Using the 6 OmpA recombi...

Embodiment 3

[0031] The prokaryotic plasmid library construction of embodiment 3 shuffling OmpA gene

[0032] After the obtained OmpA shuffled gene (ompAs) product was recovered, it was double-digested with BamHI and XhoI, and the prokaryotic expression vector pET32a was also double-digested with the same endonuclease, and then transformed into Escherichia coli BL21 competent Cells were plated on LB plates containing 60 μg / ml Amp antibiotic to obtain 1542 recombinants.

[0033] Rapid screening of positive recombinants: randomly pick shuffled recombinants, use the vector pET32a as a negative control, and screen positive recombinants by comparing the size of the plasmids. Any recombinant with a larger molecular weight than the negative control can be initially identified as a positive recombinant. Results A total of 43 positive recombinants were screened out, and a prokaryotic plasmid library targeting Vibrio with shuffled OmpA gene (ompAs) was constructed, named PompAs-SV.

[0034] For th...

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Abstract

The present invention belongs to the technical field of DNA shuffling, and specifically discloses a bacterial outer membrane protein ompAs-19 after DNA shuffling and application thereof as an immunomodulator. The invention for the first time uses the DNA shuffling technology combined with immunological research; the outer membrane proteins of OmpA vibrio alginolyticus, Vibrio parahaemolyticus, Edwardsiella tarda and Escherichia coli are research objects; DNA shuffling technology is employed to obtain the bacterial outer membrane protein ompAs-19 after DNA shuffling, and the amino acid sequence is shown as SEQ ID NO: 2. The bacterial outer membrane protein ompAs-19 after DNA shuffling has high immune protection to vibrio alginolyticus, and reaches relative immune protective rate (RPS) of 100%. In addition, the bacterial outer membrane protein ompAs-19 after DNA shuffling also shows cross immunogenicity to Edwardsiella tarda and the reaches immune protective rate (RPS) of 85.71%. The results indicate that No.19 shuffled OmpA (ompAs-19) can be used as a vaccine component.

Description

technical field [0001] The invention relates to the technical field of DNA shuffling, in particular to bacterial outer membrane protein ompAs-19 after DNA shuffling and its application as an immune regulator. Background technique [0002] In aquatic animal breeding, various chemical drugs and antibiotics are often used to control related diseases, but long-term use of drugs not only leads to more and more drug resistance of pathogenic bacteria, but also makes the hazards of drug residues increasingly apparent, seriously affecting the safety of aquatic products , so more attention is paid to immunization prevention through vaccines. At present, the main pathogenic bacteria in my country's aquaculture industry are Vibrio and Edwardsiella tarda, but there is no multivalent high-efficiency vaccine against a variety of bacteria. Therefore, the development of high-efficiency multivalent vaccines with immune protection has broad application prospects. [0003] Studies have found t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/28C07K14/195C07K14/245C12N15/31A61K39/106A61K39/02A61K39/108A61P31/04
CPCA61K39/0208A61K39/0258A61K39/107C07K14/195C07K14/245C07K14/28A61K2039/53A61K2039/54Y02A50/30
Inventor 彭宣宪李惠楚晓
Owner SUN YAT SEN UNIV
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