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Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component

A technology of VA0760, Vibrio alginolyticus, applied in the directions of application, medical preparations containing active ingredients, drug combinations, etc.

Inactive Publication Date: 2012-06-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, before the publication of the present invention, there has not been any publication or report on the immune regulation function of the Vibrio alginolyticus outer membrane protein VA0760 mentioned in this patent application

Method used

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  • Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component
  • Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component
  • Expression of vibrio alginolyticus outer membrane protein VA0760 and application thereof as vaccine component

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Cloning and identification of outer membrane protein VA0760 gene of Vibrio alginolyticus

[0014] 1. Amplification of Vibrio alginolyticus VA0760 gene

[0015] Since the full sequence of the Vibrio alginolyticus gene has not been determined, there is no report of the VA0760 gene sequence of this bacteria. In view of the fact that the complete sequence of Vibrio parahaemolyticus has been determined and published in the database GenBank (BA000031) in March 2006, we designed VA0760 gene primers for the PCR of Vibrio alginolyticus VA0760 according to the VP0760 sequence published in its database Amplify. The designed primers are: Upstream primer: 5'-ATA GGATCC ATGTCTTACCTAAAAGAAAAGCC-3'; downstream primer: 5'-CGC AAGCTT TTAGAAGTAGTATTCAAC-3'; the horizontal line shows the restriction site, the upstream primer introduces the BamHI restriction site, and the downstream primer introduces the HindIII restriction site. Using the Bacterial Genomic DNA Extraction Kit (TIANGEN...

Embodiment 2

[0022] Expression of Vibrio alginolyticus VA0760 protein

[0023] 1 Predicted amino acid sequence

[0024] The gene VA0760 is a medium-length fragment, and analyzed by DNAssist software to obtain its complete open reading frame ORF

[0025] 2 Prokaryotic expression of recombinants

[0026]Pick a single colony of the recombinant plasmid and inoculate it into 1 ml of LB liquid medium supplemented with kanamycin (50 mg / ml) at a ratio of 1:500 and culture overnight at 37°C until saturated. In LB medium, culture at 37°C for about 2h to OD 600 It is about 0.6 or so. IPTG was added to the culture to a final concentration of 1 mmol / L, and culture was continued at 37° C. for 3 h. The induced culture was taken out and centrifuged at room temperature for 1 min at high speed to collect the bacteria. At the same time, a control group was set up, and the pellet was resuspended in 100 μL of 2×SDS gel loading buffer, and heated at 100° C. for 5 minutes. 12000rpm, centrifuge for 10min. ...

Embodiment 3

[0028] Purification of Vibrio alginolyticus VA0760 Protein

[0029] The recombinant plasmids were picked and transformed into Escherichia coli BL21(DE3). According to the corresponding vector pET-28a, they were cultured overnight at 37°C in LB medium containing 100 μg / mL kanamycin. Immediately pick a single colony, add 5mL LB solution, shake overnight at 37°C, transfer to 200mL liquid LB medium containing antibiotics at a ratio of 1:100, culture at 37°C for about 2.5-3 hours, OD 600 =0.6, add IPTG (final concentration: 1 mmol / L) to the bottle for induction, shake at 200 r / min at 30°C for 3 hours. Centrifuge at 6000g for 10 minutes at 4°C to collect the cells, wash the cells twice with normal saline, and weigh the wet weight of the cells.

[0030] The harvested bacteria were dissolved in buffer D (8M urea; 0.1M NaH) at 1:100 (g / mL) 2 PO 4 ; 10mmTris-HCl, pH 8.0) to suspend the bacteria, ultrasonically break, output power 60%, 5s / time, interval 9s, ultrasonication 30min. The...

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Abstract

The invention relates to the expression of a recombined outer membrane protein VA0760 of Vibrio alginolyticus and the action function of a vaccine component. Through cloning, expressing and purifying genes of the recombined outer membrane protein VA0760 of the Vibrio alginolyticus, a purified product can obviously improve the resistance of the fish to pathogenic bacteria such as Vibrio alginolyticus, pseudomonas fluorescens. As a target candidate of a subunit vaccine, the recombined outer membrane protein VA0760 of the Vibrio alginolyticus has great significance on the immunological prevention and treatment of controlling Vibrio infection.

Description

technical field [0001] The invention relates to the immune regulation function of an outer membrane protein (outer membrane protein, OM protein) VA0760 expressed by Vibrio alginolyticus. Background technique [0002] Vibrio alginolyticus is widely distributed in seawater and estuaries all over the world, and the number ranks first among vibrio alginolyticus. For the pathogenic microorganisms of Vibrio alginolyticus, the focus of research is immunological analysis, to clarify its pathogenic mechanism and find effective The prevention and treatment methods mainly include drugs, namely antibiotics and immune prevention and treatment. Although antibiotics have made great progress in the initial use, however, with the widespread use of antibiotics, a series of related problems have also emerged. On the one hand The irrational use of antibiotics increases the emergence of antibiotic-resistant bacteria and bacterial infections; on the other hand, artificially increasing the dose du...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/28C12N15/31A61K39/106A61P37/04
Inventor 彭宣宪熊筱鹏李惠
Owner SUN YAT SEN UNIV
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