Koi herpesvirus ORF25 nucleic acid vaccine

A koi herpes virus, ORF25 technology, applied in the directions of viral peptides, antiviral agents, biochemical equipment and methods, etc., can solve the death of carp, the blow of carp breeding industry, and the inability of koi herpes virus disease to play a complete immune protection effect and other problems, to achieve the effect of small immune dose, maintenance of ecological stability, and reduction of drug use

Inactive Publication Date: 2013-03-20
JILIN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the disease is more common in carp farming in my country, causing a large number of carp deaths and bringing almost devastating blows to the carp farming industry.
Although there are a variety of traditional vaccines that can be used for immune prevention of diseases, the production practice and our experimental research results show that the use of inactivated vaccines to immunize and prevent koi herpes virus disease cannot achieve complete immune protection. There is no report on the isolation or research of KHV attenuated strain virus, so there is an urgent need for a new vaccine for this virus to come out

Method used

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  • Koi herpesvirus ORF25 nucleic acid vaccine
  • Koi herpesvirus ORF25 nucleic acid vaccine
  • Koi herpesvirus ORF25 nucleic acid vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: Cloning and identification of ORF25 gene

[0022] 1. Design ORF25 gene amplification primers

[0023] According to the KHV-I strain ORF25 gene sequence registered on GenBank, primers were designed using the software Primer Premiers 5.0, and the upstream primer was: F1 5′- GATATC ATGACGGGTTGTGGGGTT-3′, the downstream primer is: F2 5′- GAATTC TTAGGGCCTCCGGGA-3', two restriction enzyme cutting sites (underlined sites) of EcoR V and EcoR I were respectively introduced, and the primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0024] 2. PCR amplification and identification of ORF25 gene

[0025] Using KHV nucleic acid DNA as a template, the ORF25 gene was amplified by PCR. The reaction system is: F1, F2 (20pmol / μL) 0.5μL, 10×ExBuffer 2.5μL, dNTP 2μL, DNA 1μL, ExTaq enzyme 0.25μL, add sterilized water to make up to 25μL. The PCR reaction conditions were: pre-denaturation at 95°C, 5min; denaturation at 94°C, 40s; anne...

Embodiment 2

[0027] Example 2: Construction and identification of pIRES-ORF25 recombinant eukaryotic expression plasmid

[0028] 1. Preparation of pIRES-neo plasmid

[0029] Take 5 μl of DH5α bacteria liquid containing pIRES-neo plasmid (purchased from Kingvik (China) Biotechnology Center), add it to 5 mL of LB liquid medium containing ampicillin, and culture overnight at 37°C with shaking at 180r / min. A small amount of plasmids were prepared according to the SDS alkaline lysis method of the Molecular Cloning Operation Guide (Third Edition).

[0030] 2. Digestion of pIRES-neo plasmid

[0031] The pIRES-neo plasmid was subjected to EcoR V and EcoR I double enzyme digestion, 1.0% agarose electrophoresis, and a large piece of enzyme digestion was recovered according to the instructions of the Tiangen company's agarose gel DNA recovery kit.

[0032] 3. Cloning of ORF25 gene in pIRES-neo plasmid

[0033]Use Tiangen Company Agarose Gel DNA Recovery Kit to recover and purify the PCR product of...

Embodiment 3

[0037] Example 3: Expression of recombinant plasmid pIRES-ORF25 in MFC cells

[0038] 1. Mass extraction and purification of recombinant plasmids

[0039] Transform the identified positive pIRES-ORF25 plasmid into Escherichia coli DH5α competent cells, pick a single colony; inoculate it in 5 mL of LB liquid culture medium containing Amp+, culture with shaking at 37°C until the logarithmic growth phase (OD600≈0.6); Take 1mL and inoculate it into 500mL LB liquid culture solution containing Amp to expand the culture. Centrifuge the above culture at 5000r / min for 10min, discard the supernatant, place it in a low-temperature freezer for several hours, and use 200ml ice-precooled STE (0.1mol / L NaCl, 10mmol / L Tris-HCl pH8.0, 1mmol / L EDTA ) to resuspend the bacterial pellet, and collect the bacteria by centrifugation as described above.

[0040] Refer to the Molecular Cloning Operation Guide (Third Edition) SDS alkaline lysis method to prepare a large number of plasmids and extract ...

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Abstract

The invention discloses a koi herpesvirus ORF25 nucleic acid vaccine, which has good immunogenicity. According to the koi herpesvirus ORF25 nucleic acid vaccine, ORF25 gene is cloned to an eukaryotic expression vector pIRES-neo to obtain recombined plasmid. The koi herpesvirus ORF25 nucleic acid vaccine has effective prevention effect to koi herpes.

Description

technical field [0001] The invention relates to a koi herpes virus nucleic acid vaccine, which belongs to the field of biotechnology. Background technique [0002] Koi herpesvirus disease (KHVD) is a highly contagious and fatal disease caused by koi herpesvirus (KHV) in koi, carp and its common variants such as framed mirror carps. disease. [0003] The disease first broke out in the Magan Michael area of ​​Israel and the United States in 1998. In 2000, the disease spread to the United Kingdom, Germany and Belgium; in April 2002, the disease occurred in Indonesia, and the koi carp in Guangdong Province, China was suspected to have the disease; in the same year In December, Taiwan Province of my country confirmed that koi were infected with the disease; in October 2003, Japan confirmed that the disease had already existed in the country. At present, the disease is more common in carp culture in my country, causing a large number of carp deaths and bringing almost devastating...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/245A61P31/22C12N15/79C12N15/38C07K14/03
Inventor 周井祥张东鸣王好李新伟张伟祖岫杰杨春桥郭海勇
Owner JILIN AGRICULTURAL UNIV
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