CN-122012618-A - Bovine epizootic fever virus strain and reverse genetic application thereof

CN122012618ACN 122012618 ACN122012618 ACN 122012618ACN-122012618-A

Abstract

The invention belongs to the field of veterinary virology, and discloses a bovine epizootic fever virus strain and reverse genetic application thereof, wherein the reverse genetic step is to prepare plasmid for expressing complete genome of bovine epizootic fever virus by taking pCI plasmid as a framework; the pCAGGS plasmid is taken as a framework to respectively prepare auxiliary plasmids for expressing N protein genes, P protein genes, L protein genes and G protein genes of bovine epidemic fever viruses, BHK-21 cells are co-transfected by the five plasmids, and the cells are cultured to obtain the saved bovine epidemic fever viruses. The reproduction capacity of the rescued bovine epidemic heat virus is not obviously different from that of the parent virus, and the genome is still stable after 10 passages of serial passages in BHK-21 cells. The invention provides methodological support for the genetic modification of bovine epidemic fever virus and has application prospect.

Inventors

YIN XIN
WANG FANG
ZHANG MINMIN
CHANG JITAO
JIANG ZHIGANG
SUN CHAO

Assignees

中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心）

Dates

Publication Date: 20260512
Application Date: 20260413

Claims (13)

1. A plasmid combination comprising a first plasmid, a second plasmid, a third plasmid, and a fourth plasmid; the first plasmid contains a first gene expression cassette; the second plasmid contains a second gene expression cassette; The third plasmid contains a third gene expression cassette; the fourth plasmid contains a fourth gene expression cassette; The first gene expression cassette is capable of transcribing mRNA containing the coding sequence of the N protein of bovine epizootic fever virus; The second gene expression cassette is capable of transcribing mRNA containing the coding sequence of the P protein of bovine epizootic fever virus; the third gene expression cassette is capable of transcribing mRNA containing the coding sequence of the L protein of bovine epizootic fever virus; The fourth gene expression cassette is capable of transcribing mRNA containing the coding sequence of the G protein of bovine epizootic fever virus.
2. The plasmid combination of claim 1 further comprising a fifth plasmid comprising a fifth gene expression cassette capable of transcribing genomic RNA of bovine epizootic fever virus.
3. The plasmid combination of claim 2 wherein the backbone plasmid of the first plasmid is a pCAGGS plasmid; the framework plasmid of the second plasmid is pCAGGS plasmid; the backbone plasmid of the third plasmid is pCAGGS plasmid; The skeleton plasmid of the fourth plasmid is pCAGGS plasmid, or The backbone plasmid of the fifth plasmid is pCI plasmid.
4. The plasmid combination of claim 2 wherein in the first gene expression cassette, the promoter is a T7 promoter; In the second gene expression cassette, the promoter is a T7 promoter; in the third gene expression cassette, the promoter is a T7 promoter; In the fourth gene expression cassette, the promoter is T7 promoter, or In the fifth gene expression cassette, the promoter is a T7 promoter.
5. The plasmid combination of any one of claims 2-4, wherein the N protein of bovine epizootic fever virus has an amino acid sequence as shown in SEQ ID No. 4; the amino acid sequence of the P protein of the bovine epizootic fever virus is shown as SEQ ID NO. 5; the amino acid sequence of the G protein of the bovine epizootic fever virus is shown as SEQ ID NO. 7; The amino acid sequence of the L protein of the bovine epizootic fever virus is shown as SEQ ID NO.8, or The genome of the bovine epizootic fever virus is a sequence shown as SEQ ID NO.3 or a sequence shown as the T-to-C of 9185 th site of SEQ ID NO. 3.
6. A cell combination comprising a first cell, a second cell, a third cell, and a fourth cell; The first cell comprises the first plasmid of claim 1, 3, 4 or 5; the second cell comprises the second plasmid of claim 1, 3, 4 or 5; the third cell comprises the third plasmid of claim 1, 3, 4 or 5; the fourth cell contains the fourth plasmid of claim 1, 3, 4 or 5.
7. The combination of cells of claim 6, wherein the host cell of the first cell is selected from the group consisting of a BHK-21 cell, a BHK-T7 cell stably expressing a T7 RNA polymerase, and an e. The host cell of the second cell is selected from the group consisting of a BHK-21 cell, a BHK-21 cell stably expressing T7 RNA polymerase, and an E.coli cell; The host cell of the third cell is selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase and E.coli cells, or The host cell of the fourth cell is selected from the group consisting of BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and E.coli cells.
8. A cell combination comprising a first cell, a second cell, a third cell, a fourth cell, and a fifth cell; The first cell comprising the first plasmid of any one of claims 2-5; the second cell comprising the second plasmid according to any one of claims 2 to 5; the third cell comprising the third plasmid according to any one of claims 2 to 5; the fourth cell comprising the fourth plasmid according to any one of claims 2 to 5; the fifth cell contains the fifth plasmid according to any one of claims 2 to 5.
9. The combination of cells of claim 8, wherein the host cell of the first cell is selected from the group consisting of a BHK-21 cell, a BHK-T7 cell stably expressing a T7 RNA polymerase, and an e. The host cell of the second cell is selected from the group consisting of a BHK-21 cell, a BHK-21 cell stably expressing T7 RNA polymerase, and an E.coli cell; The host cell of the third cell is selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase and E.coli cells; the host cell of the fourth cell is selected from BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase and E.coli cells, or The host cell of the fifth cell is selected from the group consisting of BHK-21 cells, BHK-21 cells stably expressing T7 RNA polymerase, and E.coli cells.
10. A method for preparing bovine epidemic heat virus rescue virus, comprising the following steps: s1, co-transfecting bovine epizootic fever virus susceptible cells with the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid according to any one of claims 2 to 5 to obtain transfected cells; the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid are each capable of surviving in the bovine epizootic fever virus susceptible cells; The first gene expression cassette, the second gene expression cassette, the third gene expression cassette, the fourth gene expression cassette, and the fifth gene expression cassette are capable of being expressed in the bovine ephemeral fever virus susceptible cells; S2, culturing the transfected cells to obtain cultured cells; s3, separating the bovine epidemic hot virus rescue virus from the cultured cells or a culture medium for culturing the cultured cells.
11. The method of claim 10, wherein the bovine ephemeral fever virus-susceptible cell is a BHK cell; In S1, the molar usage ratio of the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid is 1:2-4:2-4:2-4:1-4; in S1, mixing the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid to obtain a plasmid mixture; mixing the plasmid mixture with a transfection reagent according to the weight ratio of 1:1-4, and standing for 10-30 minutes to obtain a transfection complex; Adding the transfection complex to a cultured monolayer of the bovine epizootic fever virus-susceptible cells to obtain the transfected cells; In S2, incubating at 35-39deg.C for 3-5 days with 4-6% CO 2 , or And S3, freezing and thawing the cultured cells, and centrifuging to obtain a supernatant to obtain the bovine epidemic heat virus rescue virus.
12. The method of claim 11, wherein the bovine ephemeral fever virus-susceptible cell is a BHK-21 cell stably expressing T7 RNA polymerase, wherein the promoter in the first gene expression cassette is a T7 promoter, wherein the promoter in the second gene expression cassette is a T7 promoter, wherein the promoter in the third gene expression cassette is a T7 promoter, wherein the promoter in the fourth gene expression cassette is a T7 promoter, and wherein the promoter in the fifth gene expression cassette is a T7 promoter; in S1, preparing said culture monolayer in a 6-well plate, wherein the total weight of said first plasmid, said second plasmid, said third plasmid, said fourth plasmid and said fifth plasmid in said transfection complex added per well is 1-3 μg; in S1, the cell density of the culture monolayer is 80% -90%; In S1, the culture medium of the culture monolayer is serum-free Opti-MEM culture medium, or The transfection reagent is PEI transfection reagent.
13. A bovine epidemic hot virus rescue virus produced by the production method according to any one of claims 10 to 12.

Description

Bovine epizootic fever virus strain and reverse genetic application thereof Technical Field The invention belongs to the field of veterinary virology, and relates to a bovine epidemic febrile virus strain and reverse genetics application thereof. Background Bovine epidemic fever is an acute, febrile, highly contagious disease caused by Bovine Epidemic Fever Virus (BEFV) EPHEMERAL FEVER. Mainly infects bovine animals, and has clinical characteristics of sudden high fever, dyspnea, arthralgia, mental depression and the like. Although the death rate is low, the milk yield of the dairy cows is suddenly reduced, the growth of the beef cattle is stagnated, and serious economic loss is brought to the animal husbandry. The virus belongs to the genus Rhabdoviridae, is an uncleaved single-stranded negative-strand RNA virus, has a genome length of about 14.8kb, and encodes 5 structural proteins, namely nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase macroprotein (L). The G protein is used as main virus immunogenicity protein, contains a plurality of neutralizing antigen sites, can induce organisms to generate protective humoral immunity, and is a core target for vaccine research and development. Reverse genetics is a genetic method for studying gene functions and their influence on phenotype by performing site-directed mutagenesis, insertion or deletion of genes. The technology of artificially modifying the virus genome cDNA and then saving the recombinant virus by cDNA cloning can accurately locate and modify the virus genes, and provides a core tool for analyzing the functions of the virus genes, researching the pathogenic mechanism and researching and developing novel vaccines. However, the reverse genetics technology of bovine epidemic fever virus is not mature, and the lack of stable and efficient whole genome cDNA cloning and matched auxiliary plasmid system at present limits the deep research on the gene functions and the development of novel vaccines. Therefore, the stable and efficient reverse genetic operating system of the bovine epidemic heat virus is constructed, and becomes one of key break-open ports for the research and development of the current bovine epidemic heat prevention and control technology. Disclosure of Invention Based on the problems existing in the prior art, the invention focuses on the BEFV reverse genetic system, optimizes and constructs the BEFV ML3 strain whole genome plasmid and auxiliary plasmid system, establishes a high-efficiency stable BEFV reverse genetic operation system, breaks through the bottleneck of the prior art, lays a foundation for deeply analyzing the BEFV pathogenic mechanism, researching and developing novel vaccines and antiviral drugs, and has great significance in improving the comprehensive prevention and control level of bovine epidemic heat and guaranteeing the healthy development of animal husbandry. In order to solve the problems occurring in the prior art, a first aspect of the present invention provides a plasmid combination comprising a first plasmid, a second plasmid, a third plasmid and a fourth plasmid; the first plasmid contains a first gene expression cassette; the second plasmid contains a second gene expression cassette; The third plasmid contains a third gene expression cassette; the fourth plasmid contains a fourth gene expression cassette; The first gene expression cassette is capable of transcribing mRNA containing the coding sequence of the N protein of bovine epizootic fever virus; The second gene expression cassette is capable of transcribing mRNA containing the coding sequence of the P protein of bovine epizootic fever virus; the third gene expression cassette is capable of transcribing mRNA containing the coding sequence of the L protein of bovine epizootic fever virus; The fourth gene expression cassette is capable of transcribing mRNA containing the coding sequence of the G protein of bovine epizootic fever virus. In some embodiments, the plasmid combination further comprises a fifth plasmid comprising a fifth gene expression cassette capable of transcribing genomic RNA of bovine epithermic virus (genomic RNA refers to genomic positive-strand RNA or positive-strand genomic RNA as the negative-strand RNA virus is named as genome in the form of a positive strand). The combination of plasmids comprising the specific N, P, L, G gene sequences of the first, second, third and fourth plasmids may be matched to the transcripts of the fifth plasmid comprising different genomic sequences to assemble a rescued virus whose passaging virus genetic characteristics are dependent on the genomic sequences in the fifth plasmid. Based on the research and development results of the invention, the plasmid combination of the first plasmid, the second plasmid, the third plasmid and the fourth plasmid has relatively independent application value, and the technical conception of the invention is embo