A kind of crucian carp hematopoietic organ necrosis yeast oral vaccine and its application

A vaccine and yeast technology, applied in the direction of vaccines, veterinary vaccines, chemical instruments and methods, etc., can solve the problems of weak immunogenicity and achieve good stability and high vaccine protection

Active Publication Date: 2020-12-25
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

[0006] Oral vaccines have the advantages of strong specificity, small molecular weight and high safety, but at the same time have the disadvantages of weak immunogenicity
So far, there is no report on the preparation of an oral vaccine against crucian carp hematopoietic organ necrosis by fusion expression of LTB and CyHV-2ORF25

Method used

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  • A kind of crucian carp hematopoietic organ necrosis yeast oral vaccine and its application
  • A kind of crucian carp hematopoietic organ necrosis yeast oral vaccine and its application
  • A kind of crucian carp hematopoietic organ necrosis yeast oral vaccine and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Selection of the target gene:

[0023] 1) Select three sequences of CyHV-2ORF25 coding gene (GenBank: KM200722.1), which are nucleic acid 763bp-963bp, 1318bp-1416bp, 1483bp-1590bp;

[0024] 2) Gene encoding ORF25-1 protein: Add the restriction site EcoRI (GAATTC) to the 763bp-963bp in step 1) at the 5' end, first add the TG gene at the 3' end, and then add the restriction site Point Not I (GCGGCCGC), the sequence is shown in SEQ ID NO.1;

[0025] 3) The gene encoding ORF25-2 protein: Add the restriction site EcoR I (GAATTC) to the 1318bp-1416bp in step 1) at the 5' end, first add the TG gene at the 3' end, and then add the enzyme digestion Site Not I (GCGGCCGC), the sequence is shown in SEQID NO.2;

[0026] 4) Gene encoding ORF25-3 protein: Add the restriction site EcoR I (GAATTC) to the 1483bp-1590bp in step 1) at the 5' end, first add the TG gene at the 3' end, and then add restriction enzyme digestion Site Not I (GCGGCCGC), the sequence is shown in SEQID NO.3;

...

Embodiment 2

[0040] Preparation of yeast oral vaccine against hematopoietic organ necrosis of crucian carp:

[0041] 1) After the 14 synthetic products of Example 1 and the pYD1 vector were double-digested with EcoR I and Not I, the synthetic products were respectively connected to the pYD1 vector to construct a yeast surface display plasmid, which was named in turn: pYD1- ORF25-1, pYD1-ORF25-2, pYD1-ORF25-3, pYD1-LTB, pYD1-LTB-ORF25-1(-), pYD1-LTB-ORF25-1, pYD1-LTB-ORF25-2(-), pYD1-LTB-ORF25-2, pYD1-LTB-ORF25-3(-), pYD1-LTB-ORF25-3, pYD1-LTB-ORF25(1+2), pYD1-LTB-ORF25(2+3), pYD1 -LTB-ORF25(1+3), pYD1-LTB-ORF25(1+2+3)( figure 1 ).

[0042] pYD1-LTB-ORF25 (1+2+3) was detected by double enzyme digestion with EcoR I and Not I. At the same time, detection primers JC-F: 5'-AGTAACGTTTGTCAGTAATTGC-3'; JC-R: 5'-GTCGATTTTGTTACATCTACAC-3' were used for PCR detection. The results showed that bands with the same size as predicted could be clearly seen in the gel imaging system, indicating that the...

Embodiment 3

[0058] Application of yeast oral vaccine against hematopoietic organ necrosis in crucian carp against CyHV-2:

[0059] 1) Vaccine oral immunization experiment

[0060] 480 crucian carp with a size of (12±2) cm were randomly divided into 16 groups, 30 in each group, respectively EBY100 / pYD1 experimental group, EBY100 / pYD1-ORF25-1 experimental group, EBY100 / pYD1-ORF25-2 experimental group group, EBY100 / pYD1-ORF25-3 experimental group, EBY100 / pYD1-LTB experimental group, EBY100 / pYD1-LTB-ORF25-1(-) experimental group, EBY100 / pYD1-LTB-ORF25-1 experimental group, EBY100 / pYD1 -LTB-ORF25-2(-) experimental group, EBY100 / pYD1-LTB-ORF25-2 experimental group, EBY100 / pYD1-LTB-ORF25-3(-) experimental group, EBY100 / pYD1-LTB-ORF25-3 experimental group , EBY100 / pYD1-LTB-ORF25(1+2) experimental group, EBY100 / pYD1-LTB-ORF25(2+3) experimental group, EBY100 / pYD1-LTB-ORF25(1+3) experimental group, EBY100 / pYD1- The LTB-ORF25 (1+2+3) experimental group and the feed without mixed vaccine were the bl...

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Abstract

The invention belongs to the field of aquatic animal disease immunization, and particularly discloses a crucian carp hematopoietic necrosis yeast oral vaccine and an application thereof. The oral vaccine is sacchar omyces cerevisiae expressing fusion protein LTB-ORF25, and the fusion protein LTB-ORF25 (1+3) has the amino acid sequence shown in SEQ ID NO.13. The protein is good in immunogenicity, good in stability in crucian carp bodies and high in vaccine protection capacity, the survival rate of crucian carps attacked by poison reaches 86.67%, and after the protein is made into the oral vaccine, operation is easy, and the oral vaccine is suitable for large-scale promotion.

Description

technical field [0001] The invention belongs to the field of aquatic animal disease immunization, and in particular relates to an oral vaccine for crucian carp hematopoietic organ necrosis yeast and its 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 virus (Ca rp pox herpesvirus, Cyprinid herpesvirus Ⅰ, CyHV-1) and koi herpesvirus (Koiherpesvirus, Cy prinid herpesvirus 3, CyHV-3), and belongs to the family Cyprinid herpesviridae. The whole genome sequencing and annotatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N1/19C07K19/00A61K39/245A61P31/22C12R1/865
CPCA61K39/12A61K2039/542A61K2039/545A61K2039/552A61P31/22C07K14/005C12N2710/16022C12N2710/16034
Inventor 周勇薛明洋范玉顶刘文枝江南孟彦李逸群曾令兵聂细荣
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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