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Recombinant DNA vaccine for resisting ecthyma and sheep pox and recombinant plasmid thereof

A technology of DNA vaccines and recombinant plasmids, which is applied in the field of preparation of double-gene recombinant DNA vaccines, and can solve the problems of economic losses in the sheep farming industry

Pending Publication Date: 2022-07-29
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The clinical symptoms of aphthous ulcers and sheep pox have certain similarities, and the impact of prevention and control due to incorrect differential diagnosis will cause immeasurable economic losses to the sheep industry
At present, there is still no therapeutic drug or vaccine that can be promoted on the market. Therefore, it is of great significance to develop a dual vaccine that can prevent these two diseases at the same time and is easy to produce, transport and store.

Method used

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  • Recombinant DNA vaccine for resisting ecthyma and sheep pox and recombinant plasmid thereof
  • Recombinant DNA vaccine for resisting ecthyma and sheep pox and recombinant plasmid thereof
  • Recombinant DNA vaccine for resisting ecthyma and sheep pox and recombinant plasmid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Amplification of target gene

[0046] 1. Design Primers

[0047] The first recombinant plasmid: pcDNA3.1-FLAG-B2L-P2A-P32-MYC

[0048] Using the ORFV B2L gene region of the ORFV isolate as a template, primers Primer 1 and Primer 2 were designed, primer Primer 1 was introduced into a partial vector sequence, BamH I restriction site, Kozak sequence and tag FLAG sequence, and primer Primer 2 was introduced into a self-cleaving peptide The first 40bp DNA sequence of P2A, the stop codon of B2L gene is removed, and a 1227bp FLAG-B2L-P2A fragment is expected to be obtained after amplification;

[0049] Primer 1: as shown in Seq ID NO: 3; Primer 2: as shown in Seq ID NO: 4.

[0050] Using the P32 gene region of SPPV isolate as a template, primers Primer 3 and Primer 4 were designed, primer Primer3 was introduced into the 40bp DNA sequence after the self-cutting peptide P2A, and primer Primer 4 was introduced into a partial vector sequence, Apa I restriction site and...

Embodiment 2

[0076] Example 2 Construction of eukaryotic recombinant plasmid

[0077] 1. Connection:

[0078] The FLAG-B2L-P2A-P32-MYC fragment was ligated into the pcDNA3.1(+) vector double-digested by BamHI and Apa I using the seamless cloning kit from Quanxingjin Company, and named pcDNA3.1-FLAG- B2L-P2A-P32-MYC recombinant plasmid; the B2L-P2A-P32 fragment was ligated into pcDNA3.1(+) vector double digested by Hind III and BamH I, and named as pcDNA3.1-B2L-P2A-P32 recombinant plasmid. The specific operation steps are as follows:

[0079] Ligation system (10μL system): 5μL 2×Basic Assembly Mix, 3μL PCR product, 2μL double-enzyme digested pcDNA3.1(+) vector, mix gently, react in 50℃ water bath for 15min, cool on ice for several seconds, -20℃ save.

[0080] 2. Conversion

[0081]Add 5 μL of the ligated product to 50 μL E.coli DH5α competent cells, mix gently, place on ice for 30 min, heat shock in a 42°C water bath for 30 s, then immediately transfer to ice for 2 min, add 450 μL LB me...

Embodiment 3

[0085] Example 3 Eukaryotic expression of recombinant plasmid

[0086] 293T cells were cultured in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum at 37°C, 5% CO 2 Incubator, spread 293T cells into 6-well plates one day before transfection, so that the cell confluence reaches 70-80% the next day; extract plasmid pcDNA3.1-FLAG-B2L- P2A-P32-MYC; according to the Hanheng LipoFiter lipofection instructions, the pcDNA3.1-FLAG-B2L-P2A-P32-MYC recombinant plasmid was transfected into 293T cells, and the amount of plasmid and transfection reagent was 4 μg and 6 μL, respectively , 6h after transfection, remove the stock solution, add 2mL 2% DMEM nutrient solution, continue to culture in the incubator for 48h, remove the stock solution, wash each well with 1mL PBS once, add 160μL RIPA lysis buffer and 1.6μL PPMSF (100×), keep on ice Lyse for 30min, transfer into a 2mL centrifuge tube, add 40μL SDS-PAGE protein loading buffer (5×), boil in boiling water for ...

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Abstract

The invention provides a recombinant DNA vaccine and a recombinant plasmid thereof, the DNA vaccine recombinant plasmid comprises an antigen coding sequence and a plasmid vector, the antigen coding sequence comprises an orf antigen coding sequence and a sheep pox antigen coding sequence which are connected in series, and an orf antigen and a sheep pox antigen are expressed as independent antigen proteins. The invention provides construction of a double-gene recombinant DNA (Deoxyribose Nucleic Acid) vaccine of an orf virus gene and a sheep pox virus gene and analysis of a mouse immune effect. According to the invention, an orf virus B2L (011) gene and a sheep pox virus P32 gene are selected, the two genes are connected in series by using a self-cleaving peptide P2A, and the self-cleaving peptide P2A generates self-cleaving in eukaryotic cells, so that two antigen proteins are independently expressed on an eukaryotic expression vector pcDNA3.1 (+). The recombinant DNA vaccine prepared by the invention also has a better immune effect after being used for immunizing a mouse, and can be used as a reference of a candidate vaccine.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a preparation method of a double-gene recombinant DNA vaccine of aphtha virus gene and sheep pox virus gene and an effect evaluation after immunizing mice. Background technique [0002] Sheep aphthous virus (also known as sheep infectious pustular virus, Orf virus, ORFV) belongs to the parapoxvirus genus, double-stranded DNA virus, is an epithelial zoonotic virus, mainly infects goats and sheep. The sheep farming industry causes huge economic losses; the lesions are limited to the skin, mucous membranes and oral cavity, and erythema or crusts will form. The virus particles can exist in the crusts and fall into the environment as the crusts fall off, reinfecting the host. . The virus is resistant to desiccation, so once infection in a flock occurs repeatedly, it is difficult to eradicate. [0003] Sheep pox virus (SPPV) is an enveloped double-stran...

Claims

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Application Information

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IPC IPC(8): C12N15/85A61K39/275A61P31/20
CPCC12N15/85A61K39/12A61P31/20C12N2710/24234C12N2710/24034C12N2800/107A61K2039/53A61K2039/552A61K2039/70Y02A50/30
Inventor 陆慧君文美玲赵魁王妍高飞贺文琦关继羽李姿
Owner JILIN UNIV
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