A kind of crucian carp herpes virus disease jdorf25 vaccine and its preparation method and application

A crucian carp herpes virus and vaccine technology, applied in the biological field, can solve the problems of high-efficiency expression of heterogeneous biological cells and difficulty of exogenous genes, and achieve the effects of high expression, simple cultivation, and good safety

Active Publication Date: 2017-04-12
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 kind of crucian carp herpes virus disease jdorf25 vaccine and its preparation method and application
  • A kind of crucian carp herpes virus disease jdorf25 vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Obtaining of the protein encoded by the JDORF25 gene:

[0057] 1. Truncated region screening

[0058] The antigenic region, hydrophilic region and surface display probability of ORF25 gene of carp herpesvirus type Ⅱ were analyzed, and the hydrophilic sequence with concentrated antigenic determinants and strong antigenicity was selected as the proposed expression gene.

[0059] 2. Codon optimization

[0060] 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 gene (JDORF25) 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 .

[0061] 3. Amplification of JDORF25 gene

[0062] 3.1 Design of PCR primers for JDORF25 gene

[0063] According t...

Embodiment 2

[0107] A preparation method of crucian carp herpes virus disease vaccine, the steps are as follows:

[0108] Pick the Pichia pastoris Km71 / CyHV-2-25 strain, inoculate it into 100mL BMGY medium, and culture it with shaking at 30°C and 250r / min until 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, remove the yeast in the induction solution by centrifugation (remove the precipitate and leave the supernatant), and use the Bradford protein analysis method to calculate the content of the target protein (recombinant protein JDORF25) in the original induction su...

Embodiment 3

[0112] A kind of application of crucian carp herpes virus disease vaccine in the preparation of carp herpes virus type II vaccine, its application process is as follows:

[0113] The recombinant protein JDORF25 prepared in Example 2 is used to immunize crucian carp (250 ± 20 grams) with substantially no difference in body weight, good mental condition, and carp herpes virus type II negative, and a positive control and a normal saline control (each crucian carp intramuscular injection of each group) are established at the same time. 100 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.

[0114] Table 1 Grouping of crucian carp immune protection experiment against herpes virus disease

[0115]

[0116]

...

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Abstract

The invention discloses a crucian herpesvirus disease JDORF25 vaccine as well as a preparation method and an application thereof. The crucian herpesvirus disease vaccine is recombinant protein truncated, optimized and translated by crucian herpesvirus type II immuno-related ORF25 gene, wherein the sequence is shown in SEQ ID NO.2; the gene segment is synthesized, transferred to a yeast expression vector and screened by highly copying and cloning to finally obtain a high-expression recombinant protein pichia pastoris Km71 / CyHV-2-25 Pichia pastoris Km71 / CyHV-2-25, CCTCC NO: M2014570. The protein fermented by the yeast has the characteristics of high activity and high yield. The passive immunity test defines that the antigen protein can effectively prevent crucian herpesvirus disease and can also be mixed with adjuvants for highly effectively preventing crucian herpesvirus disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a crucian carp herpes virus disease vaccine (JDORF25), a preparation method and an 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, Cyprinid herpesvirus 3, CyHV-3), and is a member of Cyprinid herpesviridae. The whole genome sequencing and annotation research of CyHV-2 has been completed, and the resear...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/38C12N1/19C07K14/03A61K39/245A61P31/22C12R1/84
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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