A nucleic acid vaccine of Chinese rainbow trout infectious hematopoietic necrosis disease and its application

A technology of hematopoietic organ necrosis and nucleic acid vaccine, applied in the field of genetic engineering, can solve the problem of no nucleic acid vaccine and other problems

Active Publication Date: 2019-08-02
HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no relevant report on how to optimize the nucleic acid vaccine

Method used

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  • A nucleic acid vaccine of Chinese rainbow trout infectious hematopoietic necrosis disease and its application
  • A nucleic acid vaccine of Chinese rainbow trout infectious hematopoietic necrosis disease and its application
  • A nucleic acid vaccine of Chinese rainbow trout infectious hematopoietic necrosis disease and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Primer Design and Synthesis

[0029] According to the comparison of multiple gene sequences of IHNV surface glycoprotein (glycoprotein, G) included in GenBank, the specific conserved segment of the surface glycoprotein G gene was selected, and the glycoprotein used to amplify J genotype IHNV was designed using Prime primer 5.0 software. Protein gene, namely SEQ ID No.1-6.

[0030] The primers for replacing the ampicillin resistance gene on the pcDNA3.1 vector were designed according to the sequence of the kanamycin gene (Neo) and the pcDNA3.1 vector, ie, SEQ ID No.7-8. The above sequences are shown in Table 1, and the primers were synthesized by Harbin Boshi Biological Company.

[0031] Table 1 is used to amplify the primer sequence of the IHNV glycoprotein G gene of J genotype

[0032]

[0033] Remarks: The italic part is the base complementary to the pcDNA vector

[0034] SEQ ID No.9:

[0035] pGflank (1682bp)

[0036]CTTTTGTGCTTTGAGACCGAACGCAACTCGCA...

Embodiment 2

[0045] Amplification and RNA preparation of embodiment 2 virus

[0046] Virus isolate IHNV HLJ-15 virus suspension was diluted 10 with cell maintenance fluid (MEM medium containing 2% FBS) -5 Take 1ml to inoculate confluent monolayer EPC cells, incubate at 15°C for 1h, discard the virus suspension, add 5mL of normal cell maintenance solution to the cell culture flask, and incubate at 15°C. When more than 80% of the cells appear cytopathic effect (cytopathic effect, CPE), the cell culture medium (ie virus suspension) is collected. Take 250 μL of the virus suspension in an RNase-free centrifuge tube. Centrifuge at 12 000 g for 5 min to remove the precipitate. Viral genomic RNA was extracted according to the instructions of SV Total RNA Isolation System. The extracted RNA was aliquoted and stored at -80°C for later use. Among them, the IHNV isolate HLJ-15 was preserved in the China Center for Type Culture Collection with the preservation number CCTCC V201622, and the preserva...

Embodiment 3

[0047] The amplification of embodiment 3 glycoprotein genes

[0048] According to the instructions of the one-step RT-PCR kit, use the primer pairs in the primer table to amplify the target gene. RT-PCR amplification program: pre-reaction at 50°C for 30min, pre-denaturation at 94°C for 5min, denaturation at 94°C for 1min, annealing at 50°C for 1min, extension at 72°C for 2min, cycle number 30, final extension at 72°C for 10min. The RT-PCR products were gel-recovered after 1% agarose gel electrophoresis, and the recovered products were ligated with the pMD19-T simple vector, and the ligated products were transformed into DH5α competent cells, and a single colony was picked and expanded for culture to extract the plasmid and carry out PCR identification. The correct plasmid was identified for sequencing.

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Abstract

The invention relates to a nucleic acid vaccine for infectious hemopoietic necrosis of Chinese rainbow trouts and application of the nucleic acid vaccine. A preparation method of the nucleic acid vaccine includes (1), designing and amplifying specific primer pairs of surface glycoprotein of genetype-J infectious hemopoietic necrosis viruses; (2), culturing IHN (infectious hemopoietic necrosis) virus isolates by virus sensitive cell lines, and extracting viral genome RNA; (3), performing one-step RT-PCR amplification on the viral genome RNA obtained in the step (2) by the primer pairs obtained in the step (1), and recycling RT-PCR amplified products after electrophoresis; (4), connecting the recycled products in the step (3) with pMD19-T simple carriers, converting connection products into DH5alpha competent cells, picking single colonies for amplification and culture, extracting plasmids, performing PCR verification, and sequencing the correct plasmids; (5), cloning open reading frame portions of the correct IHNV (infectious hemopoietic necrosis virus) isolate glycoprotein genes in the step (4) to eukaryotic expression vectors pcDNA 3.1 by the aid of BamH I and Xho I digestion points to create the nucleic acid vaccine.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a Chinese infectious hematopoietic necrosis disease nucleic acid vaccine, a preparation method and application thereof. Background technique [0002] Infectious haematopoietic necrosis (IHN) is caused by infectious haematopoietic necrosis virus (IHNV), a common acute viral infectious disease that harms salmon and trout. Since the first outbreak in the United States in the 1950s, IHNV has spread to more than ten countries including Europe, Australia, and Asia. According to different strains of virus, environmental factors, and age of fish, IHNV can cause up to 100% mortality of salmon and trout, which has caused huge economic losses to the salmon and trout farming industry in the world. [0003] IHNV belongs to the Rhabdoviridae family (Rhabdoviridae), a single-strand, negative-strand, RNA virus of the genus Novirhabdovirus, with a total genome length of about 11kb, including s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00C12N15/85C07K14/08C12N15/11A61K48/00A61K39/205A61P31/14C12R1/93
CPCA61K39/12A61K2039/53A61K2039/552C07K14/005C12N7/00C12N15/11C12N15/85C12N2310/10C12N2760/20021C12N2760/20022C12N2760/20034C12N2800/106
Inventor 徐黎明卢彤岩赵景壮刘淼曹永生
Owner HEILONGJIANG RIVER FISHERY RES INST CHINESE ACADEMY OF FISHERIES SCI
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