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Antigen fusion protein, coding gene and application thereof

A technology of fusion protein and antigen, which is applied in the field of bioengineering, can solve the problems of high fish disease mortality and achieve the effects of easy purification, high yield and good stability

Active Publication Date: 2021-11-23
NEIJIANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The high mortality of fish diseases caused by V.mimicus infection is the bottleneck restricting the development of aquaculture industry

Method used

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  • Antigen fusion protein, coding gene and application thereof
  • Antigen fusion protein, coding gene and application thereof
  • Antigen fusion protein, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of BL21(DE3)-pET30a-LamB-TloC-OmpN3 expression strain

[0036]According to the sequences of LamB, TolC, and OmpN3 genes reported in GenBank (GenBank accession numbers: Aeromonas victoria-Lamb CP012504.1:c3971056-3969743, Vibrio mimicus-TolC DQ296639.1, ompN3 KJ831564.1), using the epitope prediction online analysis website (http: / / imed.med.ucm.es / Tools / antigenic.pl) to analyze the antigenic determinant regions of the amino acids encoded by the three genes, and select the epitope rich region, LamB (base sequence from 70-450, amino acid sequence from 24-150), TolC (base sequence from 67-567, amino acid sequence from 23-189), OmpN3 (base sequence from 691-1050, amino acid sequence sequence from 231-350). And add 2 linker sequence amino acids (GGGGSGGGGS) in the middle of the fusion gene, add the design restriction enzyme site Nde I (CATATG) at the 5' end, add the design restriction enzyme site Hind III ( AAGCTT).

[0037] The codon optimization so...

Embodiment 2

[0048] Example 2 Massive expression and purification of LamB-TloC-OmpN3

[0049] In the LB liquid medium containing Kana, culture BL21(DE3)-pET30a-LamB-TloC-OmpN3 with shaking at 37°C for about 5-6 hours until the bacterial liquid OD 600 When the value reaches 0.8, add IPTG=0.2mM, induce for 16 hours at 15°C, and collect the bacteria by centrifugation. Co-culture 12L.

[0050] Use 20mM PB (pH7.2), 300mM NaCl, 20mM Imidazole containing 1% Triton X-100, 1mMDTT, 1mM PMSF to lyse the whole bacteria, centrifuge at 12000r / min for 10min at 4°C, separate the soluble part and inclusion body of the expressed protein The dissolved part of the solution was purified by nickel ion column affinity chromatography.

[0051] Supernatant purification: Equilibrate the Ni-IDA affinity chromatography column with 20mM PB (pH7.2), 300mM NaCl, 20mM imidazole buffer, after loading the supernatant, elute the target protein with the equilibration buffer of different concentrations of imidazole, and col...

Embodiment 3

[0054] Example 3 LamB-TloC-OmpN3 quality inspection

[0055] 1. Protein stability test (freeze-thaw experiment)

[0056] Take a piece of LamB-TloC-OmpN3 protein frozen at -80°C, place it in an ice-water bath for 5-10 minutes and wait for it to melt slowly. After melting, place it in a refrigerator at 4°C for 0.5 hours. normal.

[0057] 2. LamB-TloC-OmpN3 protein concentration and purity determination

[0058] Determination of protein concentration using Bradford protein concentration assay kit, with BSA as a standard, the results show that: protein concentration is 0529mg / mL. According to SDS-PAGE glue R250 staining result shows protein purity > 90% ( Figure 6 ).

[0059] 3. LamB-TloC-OmpN3 protein reactogenicity analysis

[0060] Refer to "Protein Electrophoresis Experimental Technology" written by Guo Yaojun for the operation process of WB experiment, using anti-His as the antibody, the results show that LamB-TloC-OmpN3 is fused with His and can be recognized by the An...

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Abstract

The invention discloses an antigen fusion protein as well as a coding gene and application thereof. The invention discloses an antigen fusion protein, which comprises an Aeromonas veronii LamB protein, a Vibrio mimicus TolC protein and an Edwardsiella ictaluri OmpN3, wherein the Aeromonas veronii LamB protein, the Vibrio mimicus TolC protein and the Edwardsiella ictaluri OmpN3 are connected through connecting peptides, and the amino acid sequence of the antigen fusion protein is as shown in SEQ ID NO. 1. According to the invention, a BL21 (DE3)-pET30a-LamB-TolC-OmpN3 expression strain is prepared, the antigen fusion protein containing three bacteria at the same time can be obtained by culturing the strain, and the obtained protein is easy in purification, has a high yield, is good in stability, and can be used for subsequent development of triple subunit vaccines and diagnostic kits.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an antigen fusion protein, its encoding gene and application. Background technique [0002] Aeromonas veronii (Aeromonas veronii), belonging to Aeromonas family, Aeromonas genus, is a Gram-negative short bacillus. The bacterium is an opportunistic merman-fish pathogenic bacterium and a common pathogenic bacterium of aquatic animal diseases. Mimic Vibrio (Vibrio mimicus), belonging to the family Vibrio and the genus Vibrio, is a Gram-negative Vibrio and a zoonotic disease that can infect a variety of economic fish, including: grass carp (Ctenopharyngodon idella ), yellow catfish (Pelteobagrusfulvidraco), southern catfish (Silurus meridionalis), koi (Cyprinus carpio), etc. The high mortality of fish diseases caused by V.mimicus infection is the bottleneck restricting the development of aquaculture industry. Edwardsiella ictaluri (Edwardsiella ictaluri) belongs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C07K16/12A61K39/02A61K39/106A61P31/04C12R1/19
CPCC07K14/195C07K14/28C07K14/24C12N15/70G01N33/6854G01N33/56911G01N33/56916A61K39/0208A61K39/107A61K39/025A61P31/04C12N2800/22C07K2319/21G01N2333/195G01N2333/24G01N2333/28A61K2039/70Y02A50/30
Inventor 贺扬王均覃川杰谢碧文文正勇王永明史晋绒
Owner NEIJIANG NORMAL UNIV
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