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Recombinant goatpox virus capable of carrying out coexpression on peste des petits ruminants virus H and F protein

A goat pox virus, co-expression technology, applied in the direction of antisense single-stranded RNA virus, virus, viral peptide, 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 peste des petits ruminants virus H and F protein
  • Recombinant goatpox virus capable of carrying out coexpression on peste des petits ruminants virus H and F protein
  • Recombinant goatpox virus capable of carrying out coexpression on peste des petits ruminants virus H and F protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Construction of recombinant goat pox virus transfer vector pLSEG-HF

[0023] The constructed pLSEG-HF 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 PPRV H and F 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 9 533 bp, and it is resistant to ampicillin.

Embodiment 2

[0024] Example 2. Construction of GTPV-HF-eGFP

[0025] 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. According to the ratio of adding 150 μL serum-free Opti-MEM and 6 μL Lipofectamine 2000 to each well, prepare solution I; according to the ratio of adding 150 μL serum-free Opti-MEM and 3 μg pLSEG-HF to each well, prepare solution II. 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 then the mixture of liposomes and pLSEG-HF 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 culture for 72–96 hours. Usually 24 hours after transfection, green fluorescence can be clearly observed in the cell monolayer, ...

Embodiment 3

[0026] Example 3. Purification of GTPV-HF-eGFP

[0027] GTPV-HF-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, and the next round of plaque purification was carried out, which was repeated 8 times.

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Abstract

The invention provides a recombinant transfer vector used for constructing a recombinant goatpox virus. The transfer carrier mainly comprises the left arm and the right arm of a TK gene, two reverse p7.5K promoters, a peste des petits ruminants virus H gene and F 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 is specifically knocked out, and therefore, the potential recombinant goatpox virus capable of carrying out coexpressionon peste des petits ruminants virus H and F 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 sheeppox virus co-expressing H and F proteins of Peste des petits ruminants virus. Background technique [0002] Peste des petits ruminants (PPR) is an acute or subacute infectious disease caused by infection of peste des petits ruminants virus (PPRV). Report the disease. The disease is transmitted through direct or indirect contact, with the respiratory tract and digestive tract as the main routes of infection, and the pathogen can be transmitted through eye, nose, mouth secretions and droplets. The disease is most likely to infect sheep and goats, and goats are more susceptible than sheep and newborn sheep than adult sheep, with a higher incidence rate, and the mortality rate of acute infection can reach 100%. [0003] PPRV is a member of Paramyxoviridae and Morbillivirus. The PPRV genome is a single-stranded negative-sense RN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/863C12N15/65C12N15/45
CPCC07K14/005C12N15/65C12N15/86C12N2710/24243C12N2760/18422C12N2800/107C12N2800/30
Inventor 刘拂晓李岭邹艳丽
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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