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A compound vaccine preparation for crucian carp herpes virus disease and its preparation method and application

A crucian carp herpes virus and preparation technology, applied in antiviral agents, biochemical equipment and methods, viral peptides, etc., can solve the problems of high-efficiency expression of heterogeneous biological cells, difficulty of exogenous genes, etc.

Active Publication Date: 2017-08-25
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Codon bias makes it difficult for cloned foreign genes to be expressed efficiently in heterogeneous biological cells

Method used

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  • A compound vaccine preparation for crucian carp herpes virus disease and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Acquisition of JDORF25, JDORF25C, JDORF25D gene encoded proteins:

[0069] 1. Truncated region screening

[0070] The antigenic region, hydrophilic region and surface display probability of ORF25, ORF25C, ORF25D genes of carp herpesvirus type II were analyzed, and the hydrophilic sequences with concentrated antigenic determinants and strong antigenicity were selected as the proposed expression genes.

[0071] 2. Codon optimization

[0072] According to the yeast codon preference, codon optimization was performed on the proposed expression gene sequence, and the GC content was kept moderate, and the optimized sequence was synthesized. Compared with the original sequence of the optimized ORF25, ORF25C, ORF25D gene (JDORF25, JDORF25C, JDORF25D) coding sequence, 20%-30% of the bases in the sequence are optimized and the low-frequency codons of Pichia pastoris are all replaced by high-frequency or sub-high frequency codons.

[0073] 3. Amplification of JDORF25, JDORF25C, ...

Embodiment 2

[0121] A preparation method of crucian carp herpes virus disease vaccine, the steps are as follows:

[0122] Pick Pichia pastoris Km71 / CyHV-2-25, Pichia pastoris Km71 / CyHV-2-25C, and Pichia pastoris Km71 / CyHV-2-25D strains, and inoculate them into 100mL BMGY culture medium, 30°C, 250r / min shaking culture to OD 600 When the value reaches about 6, centrifuge at room temperature at 1500g for 5min, resuspend the bacteria in 20mL of BMMY medium, transfer the obtained bacterial solution into a 100mL shake flask, seal the bottle, add 100% methanol to a final concentration of 0.5%, and store at 30°C, 250r / The culture was induced under the condition of 1 min, and methanol was added every 24 hours to maintain the concentration of methanol at 0.5%. After continuous induction for 3 days, the yeast in the induction solution was removed by centrifugation (removing the precipitate and leaving the supernatant), and the content of the target protein (recombinant protein JDORF25, JDORF25C, JD...

Embodiment 3

[0127] The application of a crucian carp herpes virus disease compound vaccine preparation in the preparation of carp herpes virus type II vaccine is as follows:

[0128] The vaccine prepared in Example 2 was used to immunize crucian carp (250 ± 20 grams) with basically no difference in body weight, good mental condition, and negative carp herpes virus type II, and a positive control and a normal saline control (100 microliters of each crucian carp intramuscularly injected, 200 microliters, 300 microliters, the specific concentration is shown in Table 1), after 15 days after boosting immunization with the same dose, each fish was intraperitoneally injected with 500 microliters of 1×10 6 . 5 TCID 50 / ml of carp herpesvirus type Ⅱ for challenge experiments to detect whether the antibodies produced are protective.

[0129] Table 1 Grouping of crucian carp herpes virus disease yeast immune protection experiment

[0130] test group Inoculum dose quantity Gr...

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Abstract

The invention discloses a compound vaccine preparation for crucian carp herpes virus disease and its preparation method and application. The applicant provides three strains of recombinant yeast: Pichia pastoris Km71 / CyHV-2-25, CCTCC NO: M2014570; Bath Pichia pastoris Km71 / CyHV‑2‑25C, CCTCC NO: M2014571; Pichia pastoris Km71 / CyHV‑2‑25D, CCTCC NO: M2014572. The protein fermented and produced by the yeast has the characteristics of high activity and large yield. The compound vaccine preparation of crucian carp herpes virus disease prepared by mixing these three proteins has been proved by immune protection experiments that the vaccine can effectively prevent crucian carp herpes virus disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a compound vaccine preparation for crucian carp herpes virus disease, a preparation method and application. Background technique [0002] Cyprinid herpesvirus Ⅱ (CyHV-2) infection of heterogeneous gibel carp is a serious viral disease emerging in recent years, which has caused significant economic losses. Carp herpesvirus type Ⅱ was first discovered in ornamental goldfish, known as "goldfish herpesvirus hematopoietic organ necrosis". Because the virus is the second herpesvirus isolated from carps, the International Committee on Taxonomy and Nomenclature of Viruses named it Cyprus herpesvirus type II (CyHV-2). CyHV-2 is closely related to Carp pox herpesvirus (Cyprinid herpesvirus Ⅰ, CyHV-1) and Koiherpesvirus (Koiherpesvirus, Cyp rinid herpesvirus 3, CyHV-3). The whole genome sequencing and annotation research of CyHV-2 has been completed, and the research on the important functional...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/245A61P31/22C12N1/19C12P21/02C07K14/03C12R1/84
Inventor 周勇曾令兵范玉顶徐进马杰
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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