CN117070560A

CN117070560A - Construction and application of bovine herpesvirus 1 type Us9 gene deletion vaccine strain

Info

Publication number: CN117070560A
Application number: CN202310884644.0A
Authority: CN
Inventors: 吴文学; 王朋朋; 田欣睿; 赵浩然
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2023-07-18
Filing date: 2023-07-18
Publication date: 2023-11-17

Abstract

The invention provides construction and application of bovine herpesvirus 1 type Us9 gene deletion vaccine strain. The invention utilizes CRISPR/Cas9 gene editing technology to construct bovine herpesvirus 1 type gene deletion attenuated strain, and the bovine herpesvirus 1 type gene deletion attenuated strain constructed by the method generates specific deletion mutation at a Us9 gene site. The pathogenicity and/or the mortality of the obtained bovine herpesvirus 1-type gene deletion attenuated strain are obviously reduced, and the replication of the virus is not obviously different, so that the bovine herpesvirus 1-type gene deletion attenuated strain has important potential application value.

Description

Construction and application of bovine herpesvirus 1 type Us9 gene deletion vaccine strain

Technical Field

The invention relates to construction and application of bovine herpesvirus 1 Us9 gene deletion vaccine strain.

Background

Bovine herpes virus type 1 (Bovine herpesvirus, bhv-1) can cause symptoms such as hyperpyrexia, inappetence, cough, rhinorrhea, conjunctivitis, etc. in cows and beef cattle, resulting in infectious rhinotracheitis in the cows. Vaccine immunization is still a key means for preventing and treating infectious bovine rhinotracheitis, and besides the common inactivated vaccine and subunit vaccine, the gene deletion vaccine is also a big hot spot of current research.

When the mammalian cells repair DNA damage, the probability of homologous recombination is far lower than that of non-homologous recombination, which leads to the phenomenon of low editing efficiency of virus genes. However, CRISPR/Cas9 gene editing technology has been applied to many fields by many researchers, since it can further improve the rescue efficiency of recombinant viruses by improving the efficiency of homologous recombination of mammalian cells.

The gene deletion technology can not only reduce the toxicity of a target strain through manual editing, but also stimulate organisms to generate more long-term effective humoral immunity and cellular immunity, and the gene deletion vaccine constructed by the technology not only can effectively provide immunity protection for animals, but also has the advantage of distinguishing wild virus infection from vaccine immunity, and is an important point for future vaccine research and development and application.

Disclosure of Invention

The invention aims to solve the technical problems of reducing the pathogenicity of bovine herpesvirus 1 type while not affecting proliferation capacity and immunogenicity, inducing better humoral and cellular immunity, and laying a foundation for the subsequent vaccine prevention and control of BHV-1.

The invention provides a method for constructing recombinant bovine herpesvirus 1, which comprises the steps of reducing the activity of Us9 protein in the bovine herpesvirus 1, reducing the content of Us9 protein in the bovine herpesvirus 1 or/and reducing the expression level of Us9 gene in the bovine herpesvirus 1 to obtain the constructed recombinant bovine herpesvirus 1.

In the above method, the Us9 protein is a protein of A1) or A2) as follows:

a1 The amino acid sequence of the polypeptide is shown as SEQ ID No.1 in a sequence table;

a2 A) a homologous protein having more than 98% identity to A1) and derived from bovine herpes virus type 1.

By identity is meant identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity. The 98% identity or more may be at least 98%, 99% or 100% identity.

In the above method, the resulting recombinant bovine herpesvirus type 1 induces less pathogenicity than the bovine herpesvirus type 1 of interest, and has no significant difference in proliferation potency from the bovine herpesvirus type 1 of interest.

In the method, the reduction of the activity of the Us9 protein in the bovine herpesvirus type 1, the reduction of the content of the Us9 protein in the bovine herpesvirus type 1 or/and the reduction of the expression amount of the Us9 gene in the bovine herpesvirus type 1 is realized by inhibiting the expression of the Us9 gene in the bovine herpesvirus type 1 or knocking out the Us9 gene in the bovine herpesvirus type 1.

In the above method, the knocking out the Us9 gene in the bovine herpesvirus of interest type 1 is achieved by using CRISPR/Cas9 gene editing technology.

The CRISPR/Cas9 gene editing technology accords with 5' -N in coding genes of Us9 ₂₀ -NGG-3 'or 5' -CCN-N ₂₀ -fragments of regular 3' sequence arrangement are target sequences; n represents any one of A, G, C and T, N ₂₀ Representing 20 consecutive deoxyribonucleotides.

In the above method, the knocking out of the Us9 gene in the bovine herpesvirus type 1 of interest is achieved using a substance which is any one of the following:

1) The substance includes a DNA molecule encoding sgRNA1, a DNA molecule encoding sgRNA2, and a donor fragment; the sgRNA1 is targeted to the upstream sgRNA of the Us9 gene, the sgRNA2 is targeted to the downstream sgRNA of the Us9 gene, and the donor fragment is a DNA fragment from which the Us9 gene is knocked out by homologous recombination;

2) A vector expressing the sgRNA1, a vector expressing the sgRNA2, and a vector containing the donor fragment.

Further, the target sequence of the sgRNA targeting the upstream of the Us9 gene is SEQ ID No.3, the target sequence of the sgRNA targeting the downstream of the Us9 gene is SEQ ID No.4, and the nucleotide sequence of the donor fragment is the above method, wherein the bovine herpesvirus type 1 is IBRV SZH strain.

Recombinant bovine herpesvirus type 1 constructed by the above method also falls within the scope of the present invention.

In order to solve the technical problems, the invention also provides application of substances in construction of recombinant bovine herpesvirus type 1, wherein the substances are substances for reducing the activity of Us9 proteins in bovine herpesvirus type 1, reducing the content of Us9 proteins in bovine herpesvirus type 1 or/and reducing the Us9 genes in bovine herpesvirus type 1.

In the above application, the substance is any one of the following B1) to B5):

b1 DNA molecules with nucleotide sequences of SEQ ID No.5, SEQ ID No.6 and SEQ ID No.2 respectively;

b2 Nucleic acid molecules encoding sgRNA1 and sgRNA 2;

b3 An expression cassette comprising B2) said nucleic acid molecule;

b4 A recombinant vector comprising the nucleic acid molecule of B2) or a recombinant vector comprising the expression cassette of B3);

b5 A recombinant microorganism comprising the nucleic acid molecule of B2), a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4).

The recombinant microorganism of the recombinant vector is recombinant bovine herpesvirus 1.

The recombinant bovine herpesvirus type 1 can be the recombinant bovine herpesvirus type 1 constructed by the method.

The invention also provides any one of the following applications:

P1, the method, the recombinant bovine herpesvirus type 1 or the application in preparing bovine herpesvirus type 1 vaccine or in preparing product for regulating and controlling bovine herpesvirus type 1 pathogenicity;

p2, the application of the substances in preparing products for regulating and controlling bovine herpesvirus type 1 pathogenicity.

The modulation of bovine herpesvirus type 1 pathogenicity may be the alleviation of clinical symptoms in the host following infection with bovine herpesvirus type 1.

The recombinant bovine herpesvirus type 1 described herein is understood to include not only first to second generation recombinant viruses but also their progeny.

The Us9 gene of BHV-1, as a non-essential gene for viral replication in vitro, mediates latent infection of the virus, and neuronal invasion function. In order to construct a Us9 gene deletion strain of BHV-1, an IBRV SZH strain obtained by earlier separation is used as a parent strain, a corresponding homology arm is designed according to a sequence published on GenBank and used for constructing a donor sequence, a GRISPR/Cas9 technology and a Cre/LoxP technology are used for editing a Us9 gene of the strain, so that a Us9 gene deletion strain is obtained, biological characteristics of the Us9 gene deletion strain are identified and evaluated, and finally a guinea pig is used as an infection model to evaluate pathogenicity of the gene deletion strain. The result shows that the invention successfully obtains a strain of BHV-1 U.S 9 ^- The strain is successfully deleted from the Us9 gene and is free from the pollution of the parent strain through PCR identification and sequencing results; biological characterization shows that it has a growth curve similar to that of the parent strain, but compared with the parent strain, BHV-1U S9 ^- The diameter of plaques produced by the strain is significantly reduced. In addition, the invention successfully constructs a guinea pig infection model of BHV-1 by comparing IBRV SZH strain with BHV-1 U.S 9 ^- Pathogenicity of the strain, and after Us9 gene deletion, the pathogenicity of the virus to guinea pigs was found to be significantly reduced. Through evaluation of immune effect, BHV-1 U.S. 9 was found ^- Can induce guinea pigs to produce neutralizing antibody levels similar to the parental strain and IFN-gamma levels. In conclusion, BHV-1 U.S. 9 developed by the invention ^- Has better safety and capability of inducing guinea pigs to generate immune response, and has potential as a gene deletion vaccine strain.

The beneficial effects of the invention are mainly as follows:

1. the parent strain used in the invention is a wild strain separated in the laboratory, and the prior research shows that the parent strain has higher immunogenicity than other strains in the laboratory.

2. The method is innovative. The invention uses CRISPR/Cas9 technology to improve the homologous recombination efficiency of viruses, uses Cre/LoxP recombinase system to delete EGFP labels, omits the step of reconstructing donor plasmids for deleting EGFP labels, saves time and simplifies operation.

3. After the Us9 gene is deleted, the mortality rate of strains to guinea pigs is obviously reduced.

Drawings

FIG. 1 shows the donor plasmid design of example 1 of the present invention.

FIG. 2 shows the amplified bands of the CMV-EGFP expression cassette of example 1 of the present invention at various steps in the construction process. Wherein A in FIG. 2 is a band obtained by amplifying EGFP gene, M in the figure is a Trans 2K DNAMaroer, + is a band of EGFP tag sequence fragment obtained by amplifying, and-is a negative control without adding a template; FIG. 2B shows the band obtained by amplifying the CMV-EGFP expression cassette, M shows the band of the CMV-EGFP expression cassette fragment obtained by the amplification of Trans 2K DNAMmarker, + shows the negative control without the template.

FIG. 3 shows the result of screening of parent strains in example 1 of the present invention.

FIG. 4 shows the construction process of donor plasmid in example 1 of the present invention. Wherein, A in FIG. 4 is a band obtained by amplifying a donor sequence, M in the figure is Trans 2K PlusIIDNAMarker, and other lanes are bands obtained by amplifying the donor sequence; FIG. 4B shows the EGFP fluorescence generation of the positive control pEGFP N1, and FIG. 4C shows the donor plasmid pUC19EGFP ⁺ EGFP fluorescence generation of (C) and panels (B and C) were shown at 200 μm scale.

FIG. 5 shows the construction of the sgRNA-Cas9 plasmid of example 1 of the present invention. FIG. 5A shows the identification result of a US9 upstream sgRNA-Cas9 positive colony, M in the drawing is a Trans 2K DNA Marker, the amplification identification result of other US9 upstream sgRNA-Cas9 positive colonies, only the amplified bands of lanes 2, 3, 9 and 10 are brighter and clearer, and the colonies of the four lanes are shown to be US9 upstream sgRNA-Cas9 positive colonies by sequencing; panel B of FIG. 5 shows the identification of a colony positive for USS 9 downstream sgRNA-Cas9, M in the figure is Trans 2K DNAMaroer, and the other lanes are all bands obtained by amplifying a colony positive for USS 9 downstream sgRNA-Cas 9.

FIG. 6 shows the CPE observations of BHV-1 of example 1 after infection with MDBK cells and Vero E6 cells, respectively, with the scale bar in the lower right hand corner of all pictures being 200. Mu.m.

FIG. 7 shows plaque observations after 72h infection of Vero E6 cells with BHV-1 in example 1 of the invention.

FIG. 8 is a tagged gene deleted virus rescue protocol of example 1 of the present invention.

FIG. 9 is a schematic representation of rescue and purification of BHV-1 strain in which the EGFP expression cassette replaces the BHV-1 US9 gene in example 1 of the present invention. Wherein, A of FIG. 9 is a fluorescent chart of the EGFP expression cassette replacing BHV-1 U.S 9 gene when the BHV-1 strain is rescued, B of FIG. 9 is a fluorescent chart of the EGFP expression cassette replacing BHV-1 U.S 9 gene after the BHV-1 strain is purified, and the scale bars at the right lower corner of the picture are 50 μm (A) and 100 μm (B) respectively.

FIG. 10 shows BHV-1 U.S. 9 in example 1 of the invention ^- /EGFP ⁺ Identification of strains. Wherein M is a Trans 5K DNA Marker; m1 is a Trans 2K DNA Marker.

FIG. 11 is a gene-deleted virus rescue protocol in example 1 of the present invention.

FIG. 12 shows BHV-1 U.S. 9 in example 1 of the invention ^- Identification of strains. M in the figure is a Trans 5K DNA Marker.

FIG. 13 shows BHV-1 U.S. 9 in example 2 of the invention ^- Identification results of biological characteristics of the strain. In the figure, the representative significance analysis results were that the difference was significant (p <0.05)。

FIG. 14 shows the results of clinical symptoms observed in guinea pigs according to example 2 of the present invention.

FIG. 15 is a result of evaluating the pathogenicity of a gene-deleted strain in example 2 of the present invention. In the figure, ns represents the significance analysis result as no significance difference.

FIG. 16 is an evaluation of the ability of a gene-deleted strain to induce an immune response in example 2 of the present invention. Wherein, A of FIG. 16 is the neutralizing antibody level and B of FIG. 16 is the IFN-gamma level. ns represents no significant difference, and x represents significant analysis results as very significant difference (p < 0.01).

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples described below are all conventional biochemical reagents, unless otherwise specified, and are commercially available.

1 cell, strain and experimental animal

Adherent MDBK (NBL-1) cells and Vero E6 cells were purchased from China veterinary medicine monitoring institute.

The suspension MDBK cells are prepared according to the method comprising the following steps: subculturing the adherent MDBK (NBL-1) with a low-serum suspension medium, and subculturing with a serum-free suspension medium to obtain cells, namely suspension MDBK cells; the low-serum suspension culture medium is a liquid culture medium with the volume content of fetal bovine serum of 3%; the serum-free suspension culture medium is a serum-free liquid culture medium (the preparation method has been patented as patent No. Wu Wenxue, wang Pengpeng, gellera diagram, infectious bovine rhinotracheitis virus and mycoplasma bovis bivalent inactivated vaccine, the preparation method thereof and the suspension MDBK cells [ P ] used in Beijing city: CN111671893B,2022-07-19 ").

IBRV SZH strain is described in non-patent documents "Liu Mengyao, zhang Qiunan, wu Wenxue, li Jinxiang. BVDV and IBRV dual real-time fluorescent quantitative PCR detection method was established. Chinese veterinary journal, 2019 (55) 11, 28-36", referred to as BHV-1 WT strain in the examples below.

Female guinea pigs were purchased from the laboratory animal farm in the sea lake area of Beijing.

All animal experiments of the invention are subjected to ethical examination (AW 30601202-2-1) of animal welfare and animal experiments of Chinese agricultural university.

2 experiment reagent

The experimental reagents in the following examples are shown in table 1:

TABLE 1 Experimental reagents

3 instrument and consumable

The instruments and consumables in the following examples are shown in Table 2:

table 2 instrument and consumable

4 preparation of solution

The solution formulation method in the following examples is as follows:

MDBK cell culture complete medium: 445mL of DMEM basal medium was taken, and 5mL of a mixture of green streptomycin (100X) and 50mL of horse serum were added thereto to obtain MDBK cell culture complete medium.

MDBK cell maintenance medium: 396mL of DMEM basal medium was taken, and 4mL of a mixture of green streptomycin (100X) and 100mL of MDBK cell culture complete medium were added thereto to obtain MDBK cell maintenance medium.

Virus purification medium: 3.75g of the autoclaved methylcellulose is added into 500mL of MDBK cell maintenance medium, and the mixture is placed in a shaking table at 4 ℃ to be uniformly mixed and dissolved for 24 hours, so as to obtain a virus purification medium.

Vero E6 cell whole medium: 445mL of DMEM basal medium was taken, and 5mL of a mixture of green streptomycin (100X) and 50mL of fetal bovine serum were added thereto to obtain Vero E6 cell whole medium.

Vero E6 cell maintenance medium: 396mL of DMEM basal medium was taken, and 4mL of a mixture of green streptomycin (100X) and 100mL of Vero E6 cell whole medium were added thereto to obtain Vero E6 cell maintenance medium.

Virus dilution: 495mL of DMEM basal medium was added with 5mL of a mixture of green streptomycin (100X) to obtain a virus dilution.

EXAMPLE 1 construction of a Us9 Gene-deleted bovine herpesvirus type 1 strain

The Us9 gene codes Us9 protein, and the amino acid sequence of the Us9 protein is shown as SEQ ID No.1 in a sequence table.

SEQ ID No.1：

MESPRSVVNENYRGADEADAAPPSPPPEGSIVSIPILELTIEDAPASAEATGTAAAAPAGRTPDANAAPGGYVPVPAADVDCYYSESDSETAGEFLIRMGRQQRRRHRRRRCMIAAALTCIGLGACAAAAAAGAVLALEVVPRP

In the embodiment, a CRISPR/Cas9 gene editing technology is adopted to carry out specific deletion on a Us9 gene of the BHV-1 WT strain.

In order to construct a donor plasmid for deleting a target gene, as shown in fig. 1, an EGFP gene is firstly connected to a pcdna3.1 (+) plasmid, then an EGFP expression cassette is constructed by using a CMV promoter on the plasmid and the EGFP gene, and LoxP sequences (for knocking out the EGFP gene on a subsequent virus deletion strain) are introduced before and after the expression cassette; meanwhile, the genome of BHV-1 WT strain is used as a template to amplify the upstream and downstream homology arms of the Us9 gene and are respectively connected to the upstream and downstream of the EGFP expression cassette, and finally the constructed donor sequence (upstream homology arm-LoxP sequence-EGFP expression cassette-LoxP sequence-downstream homology arm) is cloned and connected to the pUC19 plasmid, so that the donor plasmid for deleting the Us9 gene is obtained.

The specific operation is as follows:

1. construction of CMV-EGFP expression cassette

EGFP gene was amplified using pEGFP N1 plasmid as template and a primer pair consisting of E736F and E736R (sequence shown in Table 3, italic indicated as cleavage recognition sequence), and EGFP tag sequence fragment (736 bp) containing NheI and Xho I restriction endonuclease sites was successfully amplified, as shown in FIG. 2A.

Enzymatic tangentially to pcDNA3.1 (+) plasmid using NheI and XhoI restriction endonucleases and ligates the EGFP tag sequence fragment described above to linearity using T4 ligaseOn the PCDNA3.1 (+) plasmid, a recombinant vector pcDNA3.1+/EGFP is obtained ⁺ 。pcDNA3.1+/EGFP ⁺ The EGFP gene fragment is used for replacing a small fragment between NheI and XhoI recognition sites on the original pcDNA3.1 (+) plasmid, and other sequences are kept unchanged, so that the recombinant vector is obtained.

Recombinant vector pcDNA3.1+/EGFP ⁺ As templates, CMV-EGFP expression cassette was amplified with primer pairs consisting of CE-F and CE-R (see Table 3, underlined indicates LoxP sequence), and LoxP sequence was introduced at both ends of the expression cassette. The CMV-EGFP expression cassette fragment (1433 bp) containing LoxP sequences at both ends was successfully amplified, and the amplified product was purified and placed in a-20℃refrigerator for use as shown in FIG. 2B. The CMV-EGFP expression cassette fragments with LoxP sequences at the two ends are proved to be CMV-EGFP expression cassettes with the LoxP sequences at the two ends by gene sequencing comparison.

TABLE 3 primer sequences

Note that: the recognition sequence is indicated in italics and the LoxP sequence is indicated in underline.

2. Screening of parent strains

To screen parent strains for the construction of gene-deleted strains, laboratory-existing strains are used: after the inactivation of two strains of BHV-1 WT and BHV-1 GMC (vaccine strain), rabbits were immunized by intramuscular injection at the same dose, blood was collected two weeks after the second immunization, serum was separated, neutralizing antibodies in serum of each immunized group were measured using two strains of BHV-1 WT and BHV-1 GMC (vaccine strain) as neutralizing viruses, and the neutralizing antibody titers were calculated using the Reed-Muench method.

As shown in FIG. 3, BHV-1 WT strain induced rabbit produced neutralizing antibodies against different strains at a higher level than BHV-1 GMC strain, so BHV-1 WT strain was selected as a parent strain for subsequent gene deletion strain construction.

3. Construction of donor plasmid

To construct a donor sequence for replacement of the Us9 gene, the upstream and downstream homology arms of the Us9 gene were first amplified. Based on the gene sequence issued by AJ004801.1 in GenBank, primers of upstream and downstream homology arms of the gene Us9 (a primer pair for amplifying Us9Up arm consisting of Us9Up F and Us9Up R, a primer pair for amplifying Us9Down arm consisting of Us9Down F and Us9Down R) are designed, the sequences are shown in Table 4, the recognition sequences for digestion are indicated in italics, and LoxP sequences are indicated in underlines.

Extracting BHV-1 WT genome as a template, amplifying the genome by using a primer pair of an upstream homology arm and a downstream homology arm of a gene US9, using an amplified product obtained by amplifying a primer pair consisting of US9Up F and US9Up R as an upstream homology arm fragment (US 9Up arm), using an amplified product obtained by amplifying a primer pair consisting of US9Down F and US9Down R as a downstream homology arm fragment (US 9Down arm), and respectively purifying and recovering the amplified product to obtain the upstream homology arm fragment and the downstream homology arm fragment.

And (3) carrying out fusion amplification on the upstream homologous arm fragment, the CMV-EGFP expression cassette fragment with LoxP sequences at two ends and the downstream homologous arm fragment by using a fusion PCR method to obtain a fusion product, namely an Up arm-loxP-CMV-EGFP-loxP-Down arm fusion fragment, namely the constructed donor sequence.

Double-restriction enzyme digestion of the fusion product and pUC19 plasmid was performed using HindIII and BamHI restriction endonucleases, respectively, and the vector and donor sequences were ligated using T4 ligase to yield the recombinant vector pUC19EGFP ⁺ Namely, the donor plasmid. pUC19EGFP ⁺ The small fragment between HindIII and BamHI recognition sites on the original pUC19 plasmid is replaced by the Up arm-CMV-EGFP-Down arm fusion fragment, and other sequences on pUC19 are kept unchanged, so that a recombinant vector is obtained, which contains a donor sequence (namely, the Up arm-loxP-CMV-EGFP-loxP-Down arm fusion fragment).

To verify whether the constructed positive donor plasmid was able to successfully express the EGFP tag, the positive donor plasmid and the positive control pEGFP N1 plasmid were transfected into Vero E6 cells using JetPRIME Transfection Reagent, respectively, and the donor plasmid was evaluated by observing fluorescence generation 24-48h after transfection. Normal expression of EGFP fluorescent tag was observed (B of fig. 4 and C of fig. 4), indicating successful construction of positive donor plasmid containing Up arm-loxP-CMV-EGFP-loxP-Down arm, and storage of positive donor plasmid in-20 ℃ refrigerator for backup.

The donor plasmid is used as a template, a primer pair consisting of F and R (specific sequences are shown in Table 4) is used for amplification, the electrophoresis result of an amplification product is shown in A diagram of FIG. 4, the length of the donor sequence is 2442bp, and the sequence is sequenced, wherein the nucleotide sequence is SEQ ID No.2. A large number of linearized donor sequence fragments (the nucleotide sequence of which is SEQ ID No.2, the upstream homology arm of Us9 gene at positions 1-404, the LoxP sequence at positions 405-438, the CMV promoter at positions 439-1055, the coding gene of EGFP at positions 1103-1823, the LoxP sequence at positions 1824-1857, and the downstream homology arm of Us9 gene) were obtained by using the primer pair consisting of F and R and were stored in a refrigerator at-20℃for rescue of the virus in which the subsequent gene was deleted.

TABLE 4 homology arm primer sequences

SEQ ID No.2：

CGTGGTGGTGCCAGTTAGCGACGACGAATTTTCCCTCGACGAAGACTCTTTTGCGGATGACGACAGCGACGATGACGGGCCCGCTAGCAACCCCCCTGCGGATGCCTACGACCTCGCCGGCGCCCCAGAGCCAACTAGCGGGTTTGCGCGAGCCCCCGCCAACGGCACGCGCTCGAGTCGCTCTGGGTTCAAAGTTTGGTTTAGGGACCCGCTTGAAGACGATGCCGCGCCAGCGCGGACCCCGGCCGCACCAGATTACACCG

TGGTAGCAGCGCGACTCAAGTCCATCCTCCGCTAGGCGCCCCCCCCCCCGCGCG

CTGTGCCGTCTGACGGAAAGCACCCGCGTGTAGGGCTGCATATAAATGGAGCGC

TCACACAAAGCCTCGTGCGGCTGCTTCGAAGGCATAACTTCGTATAGCATACATT

ATACGAAGTTATACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC

ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC

CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG

TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTT

ACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCC

CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA

CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC

ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCA

CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCAC

CAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAA

ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTA

ACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGG

AGACCCAAGCTGGCTAGCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT

GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG

TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTC

ATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTG

ACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGA

CTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT

CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC

ACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA

CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCAT

GGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACA

TCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC

GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC

CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG

TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAATAACTT

CGTATAGCATACATTATACGAAGTTATGGCCCCGAGACCCCCGGCCCTGAGGCCC

TGGGGCGGGGCCCGACTGTCCCCTTCCCCCCTCCCCCCCGTCCGCCCGCGAGTA

AAGGCTGTCTAATTTTTTCCGCACGCCCGCGCCTGTCTTCTTAGGGAGGGGAAG

GAGGGGAGGGAGGGGAAGGAGGGGAGGGAGGGGAAGGAGGGGAGGGAGGG

GAAGGAGGGGAGGGAGGGGAAGGAGGGGAGGGAGGGGAAGGAGGGGAGGG

AGGGGAAGGAGGGGAGGGAGGGGAAGGAGGGGAGGGAGGGGAAGGAGGGG

AGGGAGGGGAAGGAGGGGAGGGAGGGGAAGGAGGGGAGGGAGGGGAAGGA

GGGGAGGGAGGGGAAGGAGGGGATTCGGGCCGGCCGAGGATTCGGGCCGGCC

GAGCGAGCGGGCCAAAGCTCCGGCTCCCCCTTCCCCCTCCCGCCCCGCGGGCG

AATAAAACAACTAGAACCACAAGATATAGAGAGGCAAGGCGCGCCTGCGCGCG

GCGTTTTATTTAAAAAAATATGACAAGGGCCGGGGAGAGGGCGGGAGAGGGGG

CCGCGGGGCCCCGCCCCCCTAAACTCGCTGGCGGCGCT

4. Construction of sgRNA plasmid

The CRISPR/Cas9 system is used to increase the efficiency of homologous recombination of donor sequences with viral genomes. The transcription capacity of the U6 promoter can be enhanced by using the sgRNA with G as the initiator by designing the sgRNA upstream and downstream of Us9 via the website http:// chopchop.cbu.uib.no.

The upstream (Us 9 up) sgRNA of the Us9 gene is named as sgRNA1, the target sequence of the sgRNA is shown as SEQ ID No.3 in a sequence table, and the sequence of the sgRNA1 is shown as SEQ ID No.5 in the sequence table.

The downstream (Us 9 down) sgRNA of the Us9 gene is named as sgRNA2, the target sequence of the sgRNA is shown as SEQ ID No.4 in a sequence table, and the sequence of the sgRNA2 is shown as SEQ ID No.6 in the sequence table.

SEQ ID No.3：GTCGTCAACGAAAACTATCGAGG；

SEQ ID No.4：GAGGTAGTGCCCCGGCCCTGAGG；

SEQ ID No.5：GTCGTCAACGAAAACTATCG；

SEQ ID No.6：GAGGTAGTGCCCCGGCCCTG。

The primer sequences for the sgRNA sequences are specifically shown in Table 5, wherein the sgRNA-F targeting the Us9up position is 9u-sgRNA1-F and the sgRNA-R is 9u-sgRNA1-R; the sgRNA-F targeted to the Us9down position is 9d-sgRNA2-F and the sgRNA-R is 9d-sgRNA2-R.

TABLE 5sgRNA sequences

Note that: italics indicate the cleavage recognition sequence.

Oligomerization of sgrnas was performed using the oligomerization system shown in table 6, under the following reaction conditions: 30min at 37 ℃;95 ℃ for 5min; the PCR instrument is cooled down to 25 ℃ in a gradient way, the speed is reduced by 0.1 ℃ per second, and the oligomerization product is placed in a refrigerator with the temperature of minus 20 ℃ for standby.

9u-sgRNA1-F and 9u-sgRNA1-R were oligomerized to give the sgRNA1 oligomerized fragment.

Oligomerization of 9d-sgRNA2-F and 9d-sgRNA2-R gave sgRNA2 oligomerization fragments.

Construction of a sgRNA plasmid targeting upstream of the Us9 gene ORF region:

the oligomerization product, sgRNA1 oligomerization fragment, and PX458M vector plasmid (purchased from Feng Hui organism, #br080) were digested with Bbs I restriction endonuclease and ligated using T4 ligase according to the ligation system shown in table 6, i.e., the sgRNA1 was ligated into the BdsI site in the PX458M plasmid, resulting in ligation products. And (3) converting the connection product into T1 competent cells, and screening positive cloning bacteria, wherein the screened positive cloning bacteria are called Us9 upstream sgRNA-Cas9 positive bacteria. Amplifying and culturing the screened Us9 upstream sgRNA-Cas9 positive bacteria, extracting positive plasmids by using an endotoxin-free plasmid small-extraction medium-quantity kit, and obtaining the positive plasmids named as PX458M-sgRNA1. The designed 9u-sgRNA1-F sequence (see Table 5) is used as an upstream primer, PX458M R is used as a downstream primer 9u-sgRNA1-R (see Table 5) to carry out PCR amplification on positive plasmid PX458M-sgRNA1, the result is shown as A diagram of FIG. 5, a target fragment with 453bp is amplified, and the sequencing verification shows that the sequence is correct, so that PX458M-sgRNA1 is the sgRNA plasmid for constructing the upstream of the ORF region of the targeting Us9 gene, and the plasmid is stored in a refrigerator at the temperature of minus 20 ℃ for standby.

Construction of a sgRNA plasmid targeting downstream of the Us9 gene ORF region:

the oligomerization product sgRNA2 oligomerization fragment was digested with Bbs I restriction endonuclease and PX458M vector plasmid (purchased from Feng Hui organism, #br080) and ligated using T4 ligase according to the ligation system shown in table 6, i.e. the sgRNA2 was ligated into the Bds I site in the PX458M plasmid to give ligation product. And (3) converting the connection product into T1 competent cells, and screening positive cloning bacteria, wherein the screened positive cloning bacteria are called Us9 downstream sgRNA-Cas9 positive bacteria. Amplifying and culturing the screened Us9 downstream sgRNA-Cas9 positive bacteria, extracting positive plasmids by using an endotoxin-free plasmid small-extraction medium-quantity kit, and obtaining the positive plasmids named PX458M-sgRNA2. The designed 9d-sgRNA2-F sequence (see Table 5) is used as an upstream primer, PX458M R is used as a downstream primer to carry out PCR amplification on positive plasmid PX458M-sgRNA2, the sequence determination is carried out, the result is shown in a diagram B of FIG. 5, a target fragment with 453bp is amplified, and the sequencing verification shows that the sequence is correct, so that PX458M-sgRNA2 is used as an sgRNA plasmid for constructing the downstream of the ORF region of the targeting Us9 gene, and the plasmid is placed in a refrigerator at the temperature of minus 20 ℃ for standby.

TABLE 6 oligomerization and ligation systems

5. BHV-1 infected Vero E6 cell verification

To verify whether the Vero E6 cell line can be used for BHV-1 infection and proliferation, a titer of 10 was obtained ^7.6 TCID ₅₀ Each mL of BHV-1 WT strain virus solution was infected with MDBK cells and Vero E6 cells at an MOI of 2,1,0.5,0.1,0.05, respectively, and cytopathic effects (cytopathic effect, CPE) were observed at 24h intervals after inoculation.

As shown in FIG. 6, the observed CPE results are shown in FIG. 6, MDBK cells appear more obvious CPE when the cells are infected for 24 hours, 70% of cells appear shedding, but Vero E6 cells do not appear obvious CPE until 48 hours after being infected with BHV-1 WT strain, which shows that BHV-1 WT strain can cause Vero E6 cells to generate CPE, but CPE forming time is later.

After 72h infection, the virus plaques were observed by staining with 2% concentration of crystal violet, and the MDBK cells were shed and not stained with crystal violet, as shown in FIG. 7, BHV-1 WT strain was able to produce more pronounced plaques on Vero E6 cells, although the number of plaques decreased with a decrease in MOI, but it was also shown that BHV-1 WT strain was able to infect and replicate and proliferate Vero E6 cell lines, and that Vero E6 cells could be a candidate cell line for rescuing BHV-1 gene-deleted strains.

6. Construction of BHV-1 strain with EGFP expression cassette replacing BHV-1 US9 gene

6.1 rescue of BHV-1 Strain in which the EGFP expression cassette replaces the BHV-1 US9 Gene

To obtain strains with EGFP expression cassette replacing BHV-1 U.S. 9 gene, the linearized donor sequence fragment obtained in the above step 2 and the sgRNA-cas9 plasmid obtained in the above step 3 (PX 458M-sgRNA1 and PX458M-sgRNA 2) were co-transfected into Vero E6 cells, specifically by plating Vero E6 cells in good growth condition into six well plates of cells, when the cell density reached 60% -80%, co-transfecting donor plasmids with sgRNA plasmids according to the sample-adding method shown in FIG. 8 (transfection ratio: PX458M-sgRNA1: PX458M-sgRNA2: linearized donor sequence fragment=1:1:2), performing liquid-transfer culture 4-12h after transfection, inoculating BHV-1 (with BHV-1 WT strain) at 3MOI 24h after transfection, performing gene deletion strain rescue, and then receiving 72h after inoculation. Repeating freezing and thawing for three times, centrifuging at 4000rpm for 5min, packaging supernatant, and storing at-80deg.C.

6.2 purification of BHV-1 Strain in which the EGFP expression cassette replaces the BHV-1 US9 Gene

And (3) diluting the virus liquid obtained in the step (6.1) by using a virus diluent by 10 times to obtain dilutions of each gradient. Meanwhile, MDBK cells with good growth state are paved in a six-hole plate of cells until the cell density reaches At 60% -80%, inoculating the diluted solution of each gradient into MDBK cells, adsorbing viruses for two hours, discarding supernatant, replacing with virus purification medium, and placing at 37deg.C and 5% CO ₂ CPE was observed every 24h in incubator culture, and when MDBK cells were infected for 24h, CPE was found to have green fluorescence, resulting in BHV-1 strain in which EGFP expression cassette replaced BHV-1 U.S 9 gene, as shown in FIG. 9A. The virus plaque with green fluorescence is selected and placed in 200 mu L of virus diluent, and then is subjected to 2-fold and 5-fold ratio dilution and then is subjected to the next round of purification, so that BHV-1 strain of the EGFP expression cassette for replacing BHV-1 US9 genes is screened. Continuously purifying for six generations to obtain purified virus liquid (BHV-1 U.S 9) ^- /EGFP ⁺ Strains), see FIG. 9B, for PCR identification.

6.3 identification of BHV-1 Strain in which the EGFP expression cassette replaces the BHV-1 US9 Gene

Primers shown in Table 7 were designed based on the donor sequence and BHV-1 U.S 9 gene, respectively, for BHV-1 U.S 9 ^- /EGFP ⁺ Whether EGFP expression cassette in strain successfully replaces BHV-1 US9 gene and whether pure BHV-1 US9 is obtained ^- /EGFP ⁺ The strain is subjected to PCR identification and sequencing verification, BHV-1 WT strain and Us9 donor plasmid are used as positive control, and negative control is set.

As can be seen from FIG. 10, the EGFP expression cassette was successfully amplified using the purified virus solution as a template, and also did not contain the Us9 gene, which indicates that the present invention successfully replaces the Us9 gene with the EGFP expression cassette, and the BHV-1 strain obtained by replacing the BHV-1 Us9 gene with the EGFP expression cassette was free from parental strain contamination, and was designated as BHV-1 Us9 ^- /EGFP ⁺ Strains.

Table 7 Gene-deleted strain identification primers

7. Construction of a Us9 Gene-deleted BHV-1 Strain

To make BHV-1 U.S. 9 ^- /EGFP ⁺ The EGFP label expression cassette in the strain is removed, and the LoxP sites at the two ends of the EGFP label expression cassette are sheared by using a Cre/LoxP system, so that a Us9 gene deleted BHV-1 strain is constructed and named as BHV-1 Us9 ^- Strains.

7.1、BHV-1 Us9 ^- Rescue of strains

Spreading Vero E6 cells with good growth state in a six-hole plate, when the cell density reaches 60% -80%, transfecting Cre enzyme plasmid (the plasmid is a recombinant plasmid obtained by replacing the open reading frame sequence of Cre enzyme with a small fragment between Nhe I enzyme and Xho I enzyme in pcDNA3.1+ plasmid and keeping other sequences of pcDNA3.1+ plasmid unchanged) into Vero E6 cells according to the sample adding method shown in the specification of jetPRIME transfection reagent, culturing in a liquid change mode after 4-12h, and inoculating BHV-1 US9 obtained in step 5 with 3MOI after transfection for 24h ^- /EGFP ⁺ Strains were then attenuated 72h after inoculation. Repeating freezing and thawing for three times, centrifuging at 4000rpm for 5min, packaging the virus supernatant, and storing at-80deg.C.

The Cre enzyme open reading frame sequence is as follows (1035 bp):

ATGGCCTCCAATTTACTGACCGTACACCAAAATTTGCCTGCATTACCGGTCGA

TGCAACGAGTGATGAGGTTCGCAAGAACCTGATGGACATGTTCAGGGATCGC

CAGGCGTTTTCTGAGCATACCTGGAAAATGCTTCTGTCCGTTTGCCGGTCGT

GGGCGGCATGGTGCAAGTTGAATAACCGGAAATGGTTTCCCGCAGAACCTGA

AGATGTTCGCGATTATCTTCTATATCTTCAGGCGCGCGGTCTGGCAGTAAAAA

CTATCCAGCAACATTTGGGCCAGCTAAACATGCTTCATCGTCGGTCCGGGCT

GCCACGACCAAGTGACAGCAATGCTGTTTCACTGGTTATGCGGCGGATCCGA

AAAGAAAACGTTGATGCCGGTGAACGTGCAAAACAGGCTCTAGCGTTCGAAC

GCACTGATTTCGACCAGGTTCGTTCACTCATGGAAAATAGCGATCGCTGCCA

GGATATACGTAATCTGGCATTTCTGGGGATTGCTTATAACACCCTGTTACGTA

TAGCCGAAATTGCCAGGATCAGGGTTAAAGATATCTCACGTACTGACGGTGG

GAGAATGTTAATCCATATTGGCAGAACGAAAACGCTGGTTAGCACCGCAGGT

GTAGAGAAGGCACTTAGCCTGGGGGTAACTAAACTGGTCGAGCGATGGATTT

CCGTCTCTGGTGTAGCTGATGATCCGAATAACTACCTGTTTTGCCGGGTCAG

AAAAAATGGTGTTGCCGCGCCATCTGCCACCAGCCAGCTATCAACTCGCGCC

CTGGAAGGGATTTTTGAAGCAACTCATCGATTGATTTACGGCGCTAAGGATGA

CTCTGGTCAGAGATACCTGGCCTGGTCTGGACACAGTGCCCGTGTCGGAGC

CGCGCGAGATATGGCCCGCGCTGGAGTTTCAATACCGGAGATCATGCAAGCT

GGTGGCTGGACCAATGTAAATATTGTCATGAACTATATCCGTAACCTGGATAG TGAAACAGGGGCAATGGTGCGCCTGCTGGAAGATGGCGATTAG)

7.2、BHV-1 Us9 ^- purification of strains

Diluting the virus solution obtained in the step 7.1 by using a virus diluent, spreading MDBK cells with good growth state in a six-hole cell plate, inoculating the diluent with each gradient into the MDBK cells when the cell density reaches 60% -80%, adsorbing viruses for two hours, discarding the supernatant, replacing the culture medium with a virus purification medium, and placing the culture medium in a temperature of 37 ℃ and 5% CO ₂ Culturing in a incubator, observing CPE every 24h, picking virus plaques without green fluorescence, placing the virus plaques in 200 mu L of virus diluent, sequentially diluting by 2 times and 5 times, and purifying in the next round. After five consecutive rounds of plaque purification, a strain free of fluorescent tag and having a Us9 gene deletion, i.e., candidate BHV-1 Us9, was obtained ^- Strains.

7.3、BHV-1 Us9 ^- Identification of strains

Candidate BHV-1 U.S. 9 was prepared using the donor sequence primers shown in Table 4, respectively ^- Strains (BHV-1 U.S. 9) ^- /EGFP ⁺ The strain and BHV-1 WT strain are used as positive control and negative control is arranged), whether EGFP expression cassette is successfully knocked out or not and whether pure BHV-1 US9 is obtained or not ^- The strain was identified by PCR and verified by sequencing.

As shown in fig. 12, 2442bp is a donor sequence fragment comprising: up arm-loxP-CMV-EGFP-loxP-Down arm, 1458bp is a fragment containing the Us9 gene of interest: up arm-Us9-Down arm and 989bp are upstream and downstream homology arm fragments deleted of Us9 gene: up arm-Down arm, BHV-1 U.S. 9 ^- The strain amplified a 989bp target band and contained no other bands, indicating candidate BHV-1 US9 ^- The strain is a strain BHV-1 US9 successfully saved ^- The strain has better purity, and the constructed strain has no pollution of parent strain and is called BHV-1 U.S 9 ^- Strains. BHV-1 U.S. 9 ^- The strain does not contain EGFP expression cassette and does not contain Us9 gene. BHV-1 U.S. 9 ^- The strain is a recombinant strain obtained by deleting the Us9 gene of the BHV-1 WT strain. BHV-1 U.S. 9 ^- The strain is a recombinant strain obtained by deleting and deleting the Us9 gene of the BHV-1 WT strain.

Example 2 BHV-1 US9 ^- Identification of biological properties of strains

1、BHV-1 Us9 ^- Establishment of multi-step growth curve of strain

To BHV-1 U.S. 9 ^- The biological characteristics of the strain are identified, and the multistep growth curve of the strain is statistically evaluated. Passaged MDBK cells were plated into 12-well cell culture plates (1X 10) ⁶ 0.01MOI of BHV-1 U.S. 9 was used per well) ^- (control is BHV-1 WT), inoculation, adsorption at 37 ℃ for 2 hours, removal of supernatant, washing for 1-2 times by using PBS, adding 1 mL/hole maintenance medium, virus recovery at 0h, 24h, 48h and 72h after infection, repeated freeze thawing for three times by a refrigerator at-80 ℃, centrifugation at 4000rpm for 5 minutes, taking supernatant virus liquid, measuring the titer of the virus at each time point, drawing a multi-step growth curve of the virus, and researching the influence on the dynamic growth of the strain after the Us9 gene is deleted.

The suspension MDBK cells were inoculated into 125mL cell shake flasks at 30 mL/flask and cell density reached 8X 10 ⁶ At individual/mL, the same volume of fresh medium was changed and the same volume was changed at 0.01MOI of BHV-1 U.S. 9 ^- Inoculating into suspension MDBK cells, and virus is collected at 0h, 24h, 48h, and 72h after inoculation, and simultaneously using ultrasonic cell disruption apparatus (Ningbo Xinzhi Biotech, SCIENTZ-JY 92-I)IDN) the harvested virus fluid is sonicated to lyse cells to harvest the virus, with the following procedures: the amplitude transformer is 2mm, works for 1s, is intermittent for 2s, has 20 percent (180 w) of power and works for 1min. Centrifuging the treated virus liquid at 4000rpm for 5min, collecting supernatant, measuring titer, and plotting BHV-1 U.S 9 ^- Multistep growth curves on suspended MDBK cells.

As a result, as shown in FIG. 13C, after two cells were infected with the same MOI, BHV-1 U.S. 9 was obtained ^- The strains all exhibited similar dynamic growth capacity to that of the parent strain BHV-1 WT, with no significant differences between the obtained virus titers (results not shown), indicating that deletion of the Us9 gene had no significant effect on the ability of the virus to proliferate in vitro.

2、BHV-1 Us9 ^- Determination of plaque size of strains

BHV-1 U.S. 9 ^- (BHV-1 WT strain is used as a control) virus liquid is diluted by 10 times, MDBK cells are paved in a six-hole plate for cell culture, when the cells grow to 60% -80%, 500 mu L/hole of the virus liquid with different dilutions are respectively infected, after adsorption for 2 hours, supernatant liquid is removed, a maintenance culture medium seed containing 0.75% methylcellulose and 2% horse serum is covered, after 48-72 hours after infection, the supernatant liquid is sucked and removed, PBS is used for washing twice, 1mL of 2% crystal violet is added into each hole for staining for 30 minutes, and plaque sizes of different strains under the same dilution are analyzed and recorded under a microscope.

For BHV-1 U.S. 9 ^- The observed and measured plaque sizes of the strain and the parent strain BHV-1 WT are shown in the A, B and D of FIG. 13 and 13, and the loss of the Us9 gene results in the significant reduction of the diameter of the plaque produced by the virus, which indicates that the loss of the Us9 gene affects the invasive ability of the strain and thus the diameter of the plaque produced.

3、BHV-1 Us9 ^- Virulence detection of strains

To detect the effect on the pathogenicity of the strain following deletion of the Us9 gene, BHV-1 Us9 was used ^- Guinea pigs were immunized with BHV-1 WT strain, respectively. 9 female guinea pigs weighing 250-350g individually were selected and grouped as shown in Table 8.

TABLE 8 grouping of test animals

Guinea pigs were anesthetized with isoflurane prior to challenge, and the same dose of virus was immunized by nasal drip after anesthesia (seed virus was harvested by culture using serum-free suspension culture method). The guinea pigs were monitored daily for changes in body temperature following challenge and scored for clinical symptoms developed according to the scoring criteria of table 9.

Table 9 clinical symptom score table

Pathological section was performed 14 days after challenge, nasal concha, tracheal and pulmonary lesions were monitored, and viral load was detected.

1) Guinea pig tissue sample isolation and preservation

Freshly isolated lung, trachea and turbinates were taken, clinical symptoms were observed and recorded, while tissue samples were weighed in sterilized 1.5mL centrifuge tubes (which had been previously weighed) and stored at-80 ℃ for identification of viral load.

2) Grinding of tissue samples: samples at-80℃were removed, returned to room temperature for solubilization, 2-3 beads from which RNase had been removed in advance were added, and 900. Mu.L of virus dilution was ground, tissue ground using a tissue pestle (QIAGEN TissueLyser II), and centrifuged at 8000rpm for 10min to obtain supernatants for detection of viral loads.

The results of the clinical symptoms of guinea pigs are shown in FIG. 14, and BHV-1 U.S. 9 is found by observation ^- Both the strain and the BHV-1 WT strain can cause severe conjunctivitis to the guinea pigs, so that a large amount of secretion appears in the eyes of the guinea pigs and the eyes are adhered; at the same time, respiratory symptoms of two experimental groups are mostly light sneezeMainly, only 1 is infected with BHV-1 U.S. 9 ^- The strains of guinea pigs show purulent nasal fluid, other guinea pigs show clear nasal fluid occasionally, and the duration of symptoms is shorter; at the same time, it was observed that the parental strain caused 1 guinea pig to develop the neurological symptoms of head and neck skew for 3-4 days.

By monitoring the body temperature change of guinea pigs (A of FIG. 15), BHV-1 US9 was found ^- The test group has 1 guinea pig with low fever on the 3 rd day after immunization, symptoms last for 3 days, the BHV-1 WT test group has abnormal low body temperature on the 6 th day after virus attack, 1 guinea pig is dead on the next day, the control group guinea pig has abnormal high body temperature on the 8 th day, and the body temperature tends to be normal after 3 days; BHV-1 U.S. 9 was found by comprehensive scoring of clinical symptoms in guinea pigs (C of FIG. 15) ^- The test group showed more pronounced symptoms at 5 and 13 days after infection, whereas the BHV-1 WT test group showed more severe symptoms starting from 5 days after infection and death at 6, 8 and 14 days (B of FIG. 15), and the pathological section was examined to show severe bleeding in the lung and a large amount of effusion in the chest, while BHV-1 U.S 9 was present ^- The guinea pigs in the virus attack group and the negative control group have no death phenomenon, which shows that the pathogenicity of BHV-1 WT is obviously higher than that of BHV-1 US9 ^- The method comprises the steps of carrying out a first treatment on the surface of the At the same time, the lung, trachea and turbinates of guinea pigs were quantitatively examined for viral load, and it was found that viral nucleic acid could be detected (results not shown).

4、BHV-1 Us9 ^- Evaluation of ability of strains to induce immune response

The level of neutralizing antibodies and IFN-gamma in guinea pig serum was detected as follows:

the serum neutralizing antibody detection method comprises the following steps:

1) Serum inactivation: the serum to be detected is inactivated in a water bath for 30min in a water bath kettle at 56 ℃.

2) Preparing a virus working solution for neutralization: serial multiple dilution of virus solution with virus dilution to obtain virus titer of 200TCID ₅₀ 0.1mL of the virus-neutralizing working solution.

3) Serum dilution: 10 sterilized 1.5mL clean centrifuge tubes are placed on a centrifuge tube rack in advance, 200 mu L of diluent is added into each centrifuge tube, 200 mu L of serum to be detected after heat inactivation is absorbed, 2 times ratio dilution is carried out, 10 dilutions are carried out, namely 1/2, 1/4, 1/8, 1/16, 1/32, 1/64, 1/128, 1/256, 1/512 and 1/1024 of the original serum concentration, then each dilution of serum diluent is added into a 96-well plate, 50 mu L of each well is added, and 4 wells of each dilution are repeated.

4) Virus neutralization: adding 50 μl of diluted virus-neutralizing working solution into each well, mixing, standing in 37 deg.C incubator, shaking 96-well plate every 15min to allow antibody in serum to react with virus, neutralizing for 1 hr, absorbing mixed solution, adding 200 μl of maintenance culture medium, standing at 37 deg.C, and 5% CO ₂ Culturing in an incubator, observing the results after 72 hours, counting the holes with cytopathy, and calculating the neutralizing antibody titer of serum by adopting a Reed-Muench method. In addition, a blank control group (containing no serum and no virus), a positive control group (containing only virus) and a negative control group (containing only serum) were set, each group having 4 parallel wells.

Guinea pig serum IFN-gamma levels were measured using the guinea pig interferon gamma ELISA kit (#sekg-0018) of solebao biotechnology limited.

By detecting the levels of neutralizing antibodies and IFN-gamma in guinea pig serum 7 days after infection (FIG. 16A and FIG. 16B), it was found that both strains were able to induce guinea pigs to produce the corresponding neutralizing antibodies and IFN-gamma, and that none were significantly different, which preliminarily reflects that the constructed gene-deleted strains were able to induce guinea pigs to produce similar levels of immune response as the parental strains. In addition, since guinea pigs vaccinated with the parent strain die within 14 days, the neutralizing antibodies and IFN-gamma levels produced by the guinea pigs induced by the parent strain at 14 days of challenge were not measured. In summary, after Us9 gene deletion, the pathogenicity of the strain is weakened, although the strain can still cause guinea pigs to generate obvious clinical symptoms, the mortality rate is obviously reduced, the safety is better in the aspect of vaccine application, and the strain can induce an immune response level similar to that of a parent strain, so that the strain has great potential and can be applied to gene deletion vaccine research.

The Us9 gene mediates the functional role of BHV-1 in reactivation from latent infection, and plays a vital role in the latent infection of viruses. The BHV-1 WT strain separated in the laboratory of the applicant has stronger immunogenicity than other strains, so that the BHV-1 WT strain is used as a parent strain with gene deletion, which is more beneficial to the development of vaccine viruses.

The homologous recombination method is used for replacing the target gene with the EGFP label, which is favorable for screening and purifying the subsequent deletion strain, and a large number of research results show that the expression of the EGFP label does not influence the immunogenicity and pathogenicity of the virus, and the recombinant strain containing the EGFP label not only maintains the growth and pathogenicity of the recombinant strain, but also can be used as a marker single side for the subsequent virus missing and carrier vaccine development.

Whether EGFP label can be expressed normally or not is particularly important for the selection of promoters, and different promoters have different regulation and control effects on exogenous genes. Common herpesvirus promoters include the Simian cavitation virus 40 promoter (SV 40), porcine pseudorabies virus (PRV) gG gene promoter, human foamy retrovirus promoter (LTR), human cytomegalovirus (early) promoter (CMV), and the like. The CMV promoter has a wide range of cell tropism and high transcriptional activity has been applied to the expression of a variety of foreign genes, so the present invention uses CMV as a promoter to initiate the expression of EGFP markers in the BHV-1 gene.

MDBK cells have low transfection efficiency and cannot be effectively introduced with exogenous genes, so that the screening of alternative cell lines for gene editing of MDBK cell-addictive viruses is attracting more and more attention of researchers. In terms of transfection efficiency, from HEK293T cells, BHK21 cells, vero E6 cells and BT cells (primary bovine testis cells) to MDBK cells, the transfection efficiency gradually decreases, and since BHV-1 cells cannot proliferate on HEK293T cells and BHK21 cells, vero E6 cells are finally selected for construction of gene deletion strains. Based on this, it was examined whether BHV-1 WT isolated in this experiment could infect Vero E6 cells, and it was finally found that BHV-1 WT strain could proliferate on Vero E6 cells (FIGS. 6 and 7) and could be used in the subsequent studies.

Since BHV-1 is unable to infect and proliferate in mice, this presents challenges in building its laboratory surrogate animal models. Therefore, constructing a suitable BHV-1 experimental animal model is the focus of research in the current vaccine field. The invention selects guinea pigs for constructing the isolated BHV-1 WT strain animal model and analyzing the pathogenicity of the gene deletion strain. As is clear from fig. 14 and 15, both strains were able to cause clinical symptoms to occur to different degrees in guinea pigs, and in the negative control group, the temperature rise occurred in the guinea pigs during the test, and it was assumed that the disease was caused by the stress of the feeding environment because no other clinical symptoms were observed later and no virus was detected in the viscera. In addition, all guinea pigs infected with the parent wild strain die, but the guinea pigs infected with the gene deletion strain do not die, which indicates that the pathogenicity of the strain per se is obviously reduced after the Us9 gene is deleted. The Us9 gene is not a main virulence gene, and the main function of the Us9 gene is to mediate the reactivation of the virus from a latent infection state, and the analysis reasons are probably due to the fact that the Us9 protein and envelope protein glycoprotein E (gE) together play a synergistic effect in the transportation of the virus from the trigeminal ganglion to the nasal epithelium, and the gE function is limited after the Us9 gene is deleted, and the gE gene is an important virulence gene, so that the pathogenicity of the strain is reduced, and the analysis reasons also indirectly prove that the Us9 gene deletion strain has better safety in the aspect of vaccine application.

The present invention has found that the level of neutralizing antibody against BHV-1 and IFN-. Gamma.levels (FIG. 16A and FIG. 16B) can be detected in guinea pig serum by detecting the immune response level of guinea pigs, and that the level of neutralizing antibody induced by the gene-deleted strain increases with the lapse of the immunization time (results not shown). Although the subsequent serum immune response index monitoring and observation cannot be performed because all the guinea pigs inoculated with the parent strain die, the result of the invention can reflect that the constructed gene deletion strain can induce the guinea pigs to generate the immune response level similar to that of the parent strain, and simultaneously lays an experimental foundation for subsequent vaccine immune evaluation. In conclusion, the proliferation capability of the strain is not affected after the Us9 gene is deleted, but the pathogenicity of the strain can be obviously reduced, and a better immune response can be induced, so that the Us9 gene deletion strain is further proved to have potential as a candidate vaccine strain, and a foundation can be laid for vaccine prevention and control of subsequent BHV-1.

The invention successfully constructs a BHV-1U 9 strain by using CRISPR/Cas9 technology mediated homologous recombination ^- The strain has a growth curve similar to that of a parent strain, but the invasiveness and pathogenicity of the strain are obviously reduced, and a foundation is laid for the development of subsequent gene deletion vaccines and carrier vaccines.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims

1. A method of constructing a recombinant bovine herpes virus type 1, characterized by: comprises the steps of reducing the activity of a Us9 protein in the bovine herpesvirus type 1, reducing the content of the Us9 protein in the bovine herpesvirus type 1 or/and reducing the expression level of a Us9 gene in the bovine herpesvirus type 1 to obtain the constructed recombinant bovine herpesvirus type 1.

2. The method according to claim 1, characterized in that: the Us9 protein is a protein of the following A1) or A2):

3. The method according to claim 1 or 2, characterized in that: the resulting recombinant bovine herpesvirus type 1 induces less virulence than the bovine herpesvirus type 1 of interest.

4. A method according to any one of claims 1-3, characterized in that: the reduction of the activity of a Us9 protein in the bovine herpesvirus type 1, the reduction of the content of the Us9 protein in the bovine herpesvirus type 1 or/and the reduction of the expression amount of a Us9 gene in the bovine herpesvirus type 1 is realized by inhibiting the expression of the Us9 gene in the bovine herpesvirus type 1 or knocking out the Us9 gene in the bovine herpesvirus type 1.

5. The method according to any one of claims 1-4, wherein: the knockout of the Us9 gene in the bovine herpesvirus type 1 of interest is accomplished using a substance that is any one of the following:

1) The substance includes a DNA molecule encoding sgRNA1, a DNA molecule encoding sgRNA2, and a donor fragment; the sgRNA1 is targeted to the sgRNA upstream of the Us9 gene, the sgRNA2 is targeted to the sgRNA downstream of the Us9 gene, and the donor fragment is a DNA fragment comprising a homology arm-loxP site-EGFP expression cassette-loxP site-downstream homology arm of the Us9 gene;

6. The method according to claim 5, wherein: the target sequence of the sgRNA targeting the upstream of the Us9 gene is SEQ ID No.5, the target sequence of the sgRNA targeting the downstream of the Us9 gene is SEQ ID No.6, and the nucleotide sequence of the donor fragment is SEQ ID No.2.

7. The method according to any one of claims 1-6, wherein: the herpesvirus of the target cattle type 1 is IBRV SZH strain.

8. Recombinant bovine herpesvirus type 1 constructed by the method of any one of claims 1-7.

9. The use of a substance in the construction of recombinant bovine herpes virus type 1, characterized in that: the substance is a substance which reduces the activity of a Us9 protein in bovine herpesvirus type 1, reduces the content of a Us9 protein in bovine herpesvirus type 1 or/and reduces the Us9 gene in bovine herpesvirus type 1.

10. Any of the following applications:

use of P1, the method of any one of claims 1-7, the recombinant bovine herpesvirus type 1 of claim 8 or the use of claim 9 in the preparation of a bovine herpesvirus type 1 vaccine or in the preparation of a product for modulating bovine herpesvirus type 1 virulence;

Use of P2, a substance according to claim 9 for the preparation of a product for modulating bovine herpesvirus type 1 pathogenicity.