Recombinant goatpox virus capable of carrying out coexpression on orf virus B2L and F1L protein

A goat pox virus, co-expression technology, applied in the direction of virus, viral peptide, virus/bacteriophage, etc., can solve the problem of destroying expression

Pending Publication Date: 2021-02-05
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Insertion of exogenous antigen gene into TK gene disrupts its expression but does not significantly affect the replication of recombinant GTPV

Method used

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  • Recombinant goatpox virus capable of carrying out coexpression on orf virus B2L and F1L protein
  • Recombinant goatpox virus capable of carrying out coexpression on orf virus B2L and F1L protein
  • Recombinant goatpox virus capable of carrying out coexpression on orf virus B2L and F1L protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Construction of recombinant goat pox virus transfer vector pLSEG-BF

[0022] The constructed pLSEG-BF model diagram is as follows figure 1 shown. The left arm of the TK gene (TK L ) and right arm (TK R ) are located upstream and downstream of the exogenous sequence, respectively, and the two are homologous recombination sites of GTPV; the directions of the ORFV B2L and F1L genes are opposite, the upstream is the p7.5K promoter, and the downstream is the "ATTTTTAT" early transcription termination signal ; The eGFP screening marker is transcriptionally regulated by the p11K promoter. The full length of the recombinant vector is 8 222bp, and it is resistant to ampicillin.

Embodiment 2

[0023] Example 2. Construction of GTPV-BF-eGFP

[0024] Inoculate GT cells in a six-hole cell culture plate, culture to 80-90% dense monolayer, inoculate GTPV AV41 vaccine strain, and act at 37°C for 2 hours. Prepare solution I according to the ratio of 150 μL serum-free Opti-MEM and 6 μL Lipofectamine 2000 per well; prepare solution II according to the ratio of adding 150 μL serum-free Opti-MEM and 3 μg pLSEG-BF per well. Add solution II to solution I dropwise, mix gently, and let stand at room temperature for 20 min. During this period, the virus liquid in the six-well plate was discarded, 1 mL of Opti-MEM was added to each well, and the mixture of liposomes and pLSEG-BF after incubation was added dropwise. 37°C 5% CO 2 Culture in an incubator for 6 hours, discard the transfection solution, add cell maintenance solution, and cultivate for 72–96 hours. Usually 24 hours after transfection, green fluorescence can be clearly observed in the cell monolayer, while the control w...

Embodiment 3

[0025] Example 3. Purification of GTPV-BF-eGFP

[0026] GTPV-BF-eGFP was isolated from the parent virus population by sequential plaque purification. Freeze and thaw the recombined virus solution twice, press 10 –1 to 10 –6 Serial dilutions were used to inoculate GT cells in six-well plates at 37°C in 5% CO 2 Cultivate in the incubator for 2h. Aspirate the virus liquid, add 2 mL of maintenance medium containing 1% low-melting point agarose to each well, and continue culturing for 5–7 days. During the culture process, green fluorescent plaques ( figure 2 a). Fluorescent plaques were picked out, added to 500 μL medium, frozen and thawed twice, for the next round of plaque purification, repeated 8 times in a row.

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Abstract

The invention provides a recombination transfer vector used for constructing a recombinant goatpox virus. The transfer vector mainly comprises the left arm and the right arm of a TK gene, two reversep7.5K promoters, an orf virus B2L gene and F1L gene independently regulated and controlled by the two reverse p7.5K promoters, a p11K promoter, an enhanced green fluorescence protein (eGFP) gene and two forward Loxp sequences, wherein the transfer vector and the goatpox virus generate homologous recombination in goat testicular cells so as to obtain the recombinant goatpox virus capable of expressing the eGFP, under the function of Cre enzyme, the eGFP gene and the p11K prompter are specifically knocked out, and therefore, the potential recombinant goatpox virus capable of carrying out the coexpression on the orf virus B2L and F1L protein is obtained.

Description

technical field [0001] The invention belongs to the technical field of recombinant virus construction, and in particular relates to a recombinant sheep pox virus co-expressing the B2L and F1L proteins of sheep mouth disease virus. Background technique [0002] Orf aphthousis, also known as contagious pustules, is caused by orf virus (ORFV) infection. The disease is most likely to infect lambs, goats and lambs, and it is mostly epidemic in groups. Adult sheep are also susceptible, and humans are also infected, so it is a zoonosis. Affected sheep mainly manifested as papules, pustules, ulcers and thick verrucous scabs on the skin and mucous membranes of the lips. The disease is mainly transmitted through close contact. The damaged skin and mucous membranes of sick sheep are the main route of transmission, and it can also be caused by stables or pastures contaminated by sick sheep. The prevention of this disease usually adopts attenuated vaccine immunization. [0003] ORFV i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63C07K14/065
CPCC07K14/005C12N7/00C12N2710/24021C12N2710/24222
Inventor 刘拂晓邹艳丽李岭
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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