CN118267464A - Recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, construction method and application thereof - Google Patents

Abstract

The invention provides a recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, and a construction method and application thereof, and the recombinant feline herpesvirus genetic engineering vaccine FHV delta gIgE/TK FCV VP1-FPV VP2 strain can generate specific antibodies aiming at feline calicivirus and feline parvovirus for immunized kittens, and is hopeful to be used as a vaccine candidate strain for effectively preventing and treating feline herpesvirus and feline calicivirus feline parvovirus. After the recombinant virus immunizes cats, the recombinant vector vaccine constructed by the method shows good safety, and the immunizes cats can be protected against the attack of FCV and FPV effectively. The antibody can effectively identify the constructed recombinant vector vaccine.

Description

Recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, construction method and application thereof

Technical Field

The invention belongs to the technical fields of virology genetic engineering technology and preventive veterinary medicine, in particular relates to the technical field of vaccine attenuated strain preparation, and in particular relates to a recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, a construction method and application thereof, and further discloses a construction method of the recombinant feline herpesvirus strain and application of the recombinant genetic vaccine.

Background

According to statistical data, about 7100 cats exist in towns of China, about 5200 dogs exist in dogs, and the cat economy rapidly rises in recent years and exceeds the dog economy. In the current consumption situation of pet vaccines, the cat vaccine is up to 44.6% of the vaccinated amount. Cat fever, calicivirus and cat rhinotracheitis are the most commonly occurring three infectious diseases of cats, have high infectivity and high pathogenicity, and seriously threaten the health of pet cats.

Cat parvovirus (Feline parvovir μs, FPV) is commonly known as Mao pestivirus, a common pathogen that infects felines, and belongs to the family Paramyxoviridae. After FPV infection, symptoms such as hyperpyrexia, vomiting, diarrhea, enteritis and severe reduction of leukocyte count are manifested, and not all infected cats will develop clinical symptoms, with the severity depending on the age, immune status and concurrent infection of the infected animals, with the highest morbidity and mortality occurring in kittens typically around 6 months of age. FPV is a single-stranded DNA virus, has no envelope, and has a virus particle diameter of about 20nm to 24nm and an icosahedral symmetrical structure. The genome consists of two open reading frames, encoding the nonstructural proteins (NS 1 and NS 2) and the capsid proteins (VP 1 and VP 2), respectively. Wherein the nonstructural proteins are involved in DNA replication, assembly and transport, VP2 in the structural proteins accounts for 90% of the virions, while VP2 proteins can stimulate the production of neutralizing antibodies and mutations in their key amino acid sites affect antigen properties and host range. FPV was first discovered by French scientists in 1928 and later successfully isolated from a variety of wild and housed carnivores such as cats, raccoons, monkeys, etc. In 1984, the first cat leukopenia virus was isolated from natural cases in China for the first time, and then the existence of the virus was found in various areas of China. The current vaccines for preventing FPV infection are two types of attenuated vaccines and inactivated vaccines. Along with the co-evolution of viruses and hosts, FPV has a variation phenomenon and cat immune failure phenomenon frequently occurs clinically, and certain difficulty is brought to the prevention and control of the disease.

The feline calicivirus (Feline calicivirus, FCV) is a pathogen causing oral and upper respiratory diseases in felines, and has the characteristics of multiple occurrence, high prevalence, high morbidity, strong infectivity, relatively low mortality, and the like. Since the first isolation of FCV in new zealand in 1957, the virus has been found in different feline species both at home and abroad. Although FCV has a relatively low mortality rate, such highly lethal variant strains have frequently appeared in recent years since the discovery in 1998 of virulent strain VSD-FCV, which can cause systemic malignant systemic disease (Virulent SYSTEMIC DISEASE, VSD) in the united states. In 2018, china reported a VS-FCV isolate for the first time, which indicates that FCV has a significant variation in China and a super-strong strain is generated. These strains can even infect adult cats vaccinated with FCV, causing systemic malignant diseases with symptoms including acute hyperthermia, oedema, skin ulcers and viremia, with mortality rates of over 50%. The genome of FCV is a single-stranded positive strand RNA molecule in which ORF2 encoding the capsid protein comprises conserved and variable sequences. Wherein the nucleotide sequences of the C and E domains are relatively easy to mutate, and in particular the encoded product of the E domain comprises an antigenic linear epitope and an amino acid mutation site characteristic of identifying VS-FCV. The E region is responsible for cellular receptor binding and immune recognition, and its destruction can lead to oral and skin ulcers. The E functional region is divided into a 5' hypervariable region and a 3' hypervariable region, with 5' HVR E being the target of immune evasion. Although some differences were found in VS-FCV, clear differences in the genetic information of the VP1 region remain to be studied for classical FCV strains and VS-FCV. At present, the prevention and control of FCV are mainly vaccination, immune antibodies existing in cat groups, maternal antibodies or antibodies generated by artificial vaccination, so that the clinical morbidity of FCV can be reduced. However, vaccinated cat populations are not fully protected from FCV infection and these populations may become recessive carriers with continued detoxification.

The cat rhinotracheitis disease (FELINE VIRAL rhinotracheitis, FVR) is commonly known as cat nose branch, and can cause serious upper respiratory disease (Upper Respiratory TRACT DISEASES, URTD) in most adult cats and kittens. Feline herpesvirus type I (Feline herpesvirus-1, FHV-1) is the primary causative agent of feline rhinotracheitis. FHV-1 can be infected by cats of all ages, but young cats of 2-4 months are most susceptible, the morbidity is 100%, and the mortality is 50%; symptoms after infection with FHV-1 include conjunctivitis, runny eyes, runny nose, coughing, sneezing, loss of appetite, and the like. FHV-1 is latent in the trigeminal ganglion after primary infection, and the virus is activated periodically under stress or hypoimmunity; secondary infection occurs in the reactivated viral carrier. FHV-1 can be co-infected with other upper respiratory pathogens, such as Chlamydia felis and FCV; simultaneous infection with FCV or chlamydia can increase the severity of clinical symptoms and extend the course of clinical disease, ultimately leading to chronic conjunctivitis. After the isolation of FHV-1 from the United states in the last century, the virus was prevalent worldwide and infection with FHV-1 occurred in a variety of other felines, including tigers, leopards, lions, and the like. FHV-1, the main pathogen of upper respiratory tract infection and eye injury, seriously affects the health of pet cats and other cats. Currently, FHV-1 vaccines suitable for use in companion clinical settings mainly include inactivated vaccines and modified attenuated vaccines (MLV), and are often used in combination with FCV vaccines.

Cat rhinotracheitis, caliciviropathy, and leukopenia (cat triad) are the most central vaccines for preventing infectious diseases in pet cats. At present, the domestic pet cat vaccine market is mainly dominated by foreign cat triple Miaosan Duoduo. The most wonderful three are approved and registered by the agricultural rural department in 2011, are the only cat-specific vaccine for obtaining formal wholesale of the agricultural rural department in China at present, and are in charge of the market in China for 13 years. But the strains of the Miaosan multi-vaccine are foreign epidemic strains, the protection rate of the domestic epidemic strains is lower, and particularly the immunity efficacy to FCV and FHV is poor and the immunity duration is short. In recent years, the novel VS-FCV strain popular in China has obvious variation and super-strong strain appears. The VS-FCV strain can even infect adult cats which are vaccinated with FCV, and after being infected by the strain, the strain can cause systemic malignant systemic diseases of the cats, and the death rate is as high as more than 50%. Traditional FCV strain vaccines have failed to achieve immunoprotection levels.

With the vigorous development of DNA recombination technology, gene editing technology and the like, vaccine research gradually transits to the direction of genetic engineering vaccines, and recombinant live vector vaccines are receiving more and more attention. The virus vaccine vectors mainly related in the current research for developing virus live vector vaccines comprise poxviruses, herpesviruses, adenoviruses and the like. FHV-1 has been the subject of choice for research into feline vector vaccines since 1990. The gI and gE glycoproteins of FHV-1 are capable of forming heterodimers and play a role in the intercellular transmission of viruses and in the transmission of infections throughout the host nervous system. When part of the gI and gE genes are deleted, the pathogenicity of the virus is significantly reduced. The TK gene of FHV-1 codes for thymus kinase and has no effect on viral replication itself. Thymus kinase produced by host cells during replication growth is utilized by the virus to complete viral proliferation. Lack of TK gene will not affect the replication and proliferation of virus in cell, but will affect the neurotropic process after FHV-1 infection, and reduce virus virulence. Multiple gene loci can be edited simultaneously by CRISPR/Cas9 mediated genome editing technology, so that the recombination success rate is improved and the test period is shortened. The simultaneous deletion of the gI, gE and TK genes was chosen to be a more efficient vaccine strategy.

By taking the feline herpesvirus as a vector, a recombinant feline herpesvirus live vector vaccine capable of expressing FCV VP1 and FPV VP2 proteins is constructed, and the policy fully utilizes the immunogenicity and safety of the feline herpesvirus. The method is hopeful to realize the prevention and treatment of three viruses through one-time inoculation, and can provide a more convenient and economic epidemic prevention means compared with the traditional triple inactivated vaccine for cats. The feline herpesvirus is taken as a vector, is expected to induce the organism to generate extensive humoral immunity, cellular immunity and mucosal immunity, and can enhance the comprehensive defenses of the immune system against FHV, FCV and FPV. The overall immune effect is expected to provide longer immune protection, reducing the frequency and cost pressure of the pet owners in vaccination. Domestic clinic mainly depends on foreign imported cat triple Miaosan multi vaccine, and independently developed vaccine is expected to reduce dependence on imported products and improve independent innovation level of China in the field of pet medicine. The method has positive promotion effect on sustainable development of domestic pet medical industry and improvement of international competitiveness.

Disclosure of Invention

The invention improves the exogenous gene capacity, exogenous protein expression level and antigen presenting efficiency of the cat herpesvirus virulence gene through the CRISPR/Cas9 gene editing technology in the early stage and the deletion of the cat herpesvirus virulence gene, and the constructed gI, gE and TK three-gene deletion virus has good safety and effectiveness for kittens. The foreign genes of the domestic epidemic VSD-FCV feline calicivirus and feline parvovirus are selected to construct the recombinant feline herpesvirus, and after immunization of cats, the constructed recombinant vector vaccine can protect the immunized cats from being attacked by the feline calicivirus and the feline parvovirus virulence respectively. The feline herpesvirus live vector vaccine of the invention inserts exogenous target genes into vector genome by using CRISPR/Cas9 mediated genome editing technology, obtains high-efficiency expression, does not influence the replication of original strains, can induce humoral immunity, cellular immunity and mucosal immunity of organisms, effectively avoids some defects of the traditional live vaccine and inactivated vaccine, and achieves the effect of preventing three diseases by one needle. Successful implementation of the project can promote the research level of domestic cat vaccines and lay a foundation for future pet vaccine research and development. This not only helps to increase the health level of the pet, but also will provide valuable experience and technical accumulation for the scientific researchers in the relevant arts.

Specifically, the invention firstly provides a recombinant feline herpesvirus strain for expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, wherein the recombinant feline herpesvirus strain is prepared by deleting gE, gI and TK genes of feline herpesvirus with the preservation number of V202259, inserting FCV antigen genes into the deleted gI/gE genes and inserting FPV antigen genes into the deleted TK genes;

Preferably, the feline herpesvirus deleted for the gI, gE and TK genes in the feline herpesvirus is FHV delta gIgE/TK eGFP-mCherry strain, which is constructed by our laboratory to be preserved and carried out (preservation number: V202260, preservation in China center for type culture Collection; preservation address: chinese Wuhan university, post code 430072; preservation date: 2022, 7 month 26).

Further, the nucleotide sequences of the FCV antigen gene and the FPV antigen gene are shown in SEQ ID NO: 1-2. The sequence is correspondingly optimized on the original sequence, and compared with the wild type, the method has the advantage that the immunogenicity of the protein is enhanced by changing the antigen gene to match codons of the feline herpesvirus gene instead of optimized codons in the traditional sense.

Further, the recombinant feline herpesvirus strain expressing the feline calicivirus VP1 protein and the feline parvovirus VP2 protein is FHV delta gIgE/TK FCV VP1-FPV VP2, and the strain is submitted to China center for preservation, and the preservation information is as follows: the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 8 and 8 days, and the preservation number is CCTCC NO: V202383.

In another aspect, the invention provides a method for preparing a recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein, comprising:

(1) Constructing a recombinant vector, designing a primer to amplify left and right recombinant arms of homologous recombination, a CMV promoter-FCV VP1 gene fragment and an SV40 polyA signal sequence fragment, and sequentially assembling the left recombinant arm-CMV-FCV VP1-SV40 polyA-right recombinant arm to obtain a recombinant transfer vector;

(2) Constructing a recombinant vector, designing a primer to amplify left and right recombinant arms of homologous recombination, a CMV promoter-FPV VP2 gene fragment and a bGH polyA signal sequence fragment, and sequentially assembling the left recombinant arm-CMV-FPV VP2-bGH polyA-right recombinant arm to obtain a recombinant transfer vector;

(3) The design provides a sgRNA combination for editing eGFP and mCherry comprising the target sequence FHV Δ gIgE/tkegfp-MCHERRY SGRNA, comprising the sequence of SEQ ID NO:3-SEQ ID NO:6, a nucleotide sequence shown in the specification;

(4) After co-transfecting the cells in the step (1), 2) and (3), infecting the cells with FHV delta gIgE/TK eGFP-mCherry strain to obtain the recombinant feline herpesvirus which does not contain eGFP and mCherry fluorescent markers, wherein the recombinant feline herpesvirus is the recombinant feline herpesvirus genetic engineering vaccine FHV delta gIgE/TK FCV VP1-FPV VP2.

In another aspect, the invention further provides a biological material comprising the recombinant feline herpesvirus strain expressing the feline calicivirus VP1 protein and the feline parvovirus VP2 protein described above, which biological material is an expression cassette, a vector, or a transgenic cell.

In another aspect, the present invention provides a genetically engineered vaccine comprising a recombinant feline herpesvirus strain expressing the feline calicivirus VP1 protein and the feline parvovirus VP2 protein;

the vaccine is a live vaccine, and is suitable for nasal drip and injection inoculation.

In another aspect, the invention provides the use of a recombinant feline herpesvirus strain expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein for the preparation of a genetically engineered vaccine for the treatment and/or prevention of diseases caused by feline herpesvirus, feline calicivirus and feline parvovirus.

Furthermore, the genetic engineering vaccine consists of an antigen and a protective agent, wherein the antigen comprises the recombinant feline herpesvirus strain.

In another aspect, the present invention provides an immunogenic composition comprising a recombinant feline herpesvirus vaccine as described above that expresses feline calicivirus VP1 protein and feline parvovirus VP2 protein; and, a pharmaceutically acceptable carrier.

Further, the pharmaceutically acceptable carrier comprises any one or more than two of MONTANIDE ISA 206, MONTANIDE ISA 201, MONTANIDE GEL 01ST, aluminum hydroxide GEL adjuvant, alum, freund's adjuvant, lipopolysaccharide, cholesterol, vegetable oil, and cytokine; preferably an aluminium hydroxide gel adjuvant.

Furthermore, the invention provides an identification antibody capable of identifying the recombinant feline herpesvirus genetic engineering vaccine expressing the feline calicivirus VP1 protein and the feline parvovirus VP2 protein, which is characterized in that the antibody is secreted by a Hybridoma cell line SHVP, and the Hybridoma cell line SHVP (hybrid mia CELLLINE SHVP 12) is preserved in China center for type culture collection; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 10 and 11 days, and the preservation number is CCTCC NO: C2023313.

Advantageous effects

The invention takes a candidate strain FHV delta gIgE/TK eGFP-mCherry of a feline herpesvirus attenuated vaccine as a vector, inserts FCV antigen genes into gI and gE genome positions through homology, and inserts the FPV antigen genes into the TK genome positions to obtain recombinant virus FHV delta gIgE/TK FCV VP1-FPV VP2 strain. The result shows that the growth curve of the recombinant strain is basically consistent with that of the corresponding parent strain, and the difference of the virus titer is not great; notably, chimeric FCV VP1 and FPV VP2 capsid proteins expressed in FHV Delta gIgE/TK FCV VP1-FPV VP2 infected CRFK cells and can self-assemble into VLPs. These results indicate that the chimeric capsid proteins are functionally and antigenically intact. In addition, VLPs exhibit a high density of repetitive epitopes on the surface, a strong immunostimulatory molecule that can be readily recognized by the immune system. The recombinant vaccine is subjected to safety and immune efficacy tests, has good safety, can induce good cellular immunity and humoral immunity response channels simultaneously, can protect cats from being attacked by virulent FCV and FPV, and can effectively relieve clinical symptoms of young cats. The recombinant feline herpesvirus genetic engineering vaccine FHV delta gIgE/TK FCV VP1-FPV VP2 strain can generate specific antibodies aiming at feline calicivirus and feline parvovirus, and is hopeful to be used as a vaccine candidate strain for effectively preventing and treating feline herpesvirus and feline calicivirus and feline parvovirus. The recombinant herpesvirus of the strain with three deletion of gI, gE and TK genes is taken as a skeleton, so that the recombinant feline herpesvirus (FHV delta gIgE/TK FCV VP1-FPV VP 2) capable of simultaneously expressing FCV VP1 and FPV VP2 is constructed, and the research result shows that the recombinant herpesvirus has good genetic stability; the immune efficacy test result also shows that the recombinant virus shows good safety after immunizing cats, and the constructed recombinant vector vaccine can protect the immunized cats from being attacked by FCV and FPV effectively. The antibody can effectively identify the constructed recombinant vector vaccine.

Drawings

Fig. 1: schematic representation of recombinant plasmids;

Fig. 2: one-step growth curves of FHV Delta gIgE/TK FCV VP1-FPV VP2 and FHV Delta gIgE/TK eGFP-mCherry;

fig. 3: FHV delta gIgE/TK FCV VP1-FPV VP2 virus electron microscope identification result;

fig. 4: the WB identification result of FHV delta gIgE/TK FCV VP1-FPV VP2 virus;

fig. 5: results of 14 days of body temperature change after toxin challenge in the immune group and the control group;

fig. 6: clinical scoring results 14 days after challenge in the immunized and control groups.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The cat herpesvirus deletion gI, gE and TK genes FHV delta gIgE/TK eGFP-mCherry strain used in the invention is constructed and stored by the laboratory of China (the storage number: V202260, the storage in China center for type culture Collection; the storage address: wuhan university, post code 430072, the storage date: 2022, 7 months and 26 days).

Example 1: construction of sgRNA expression vectors

Referring to eGFP and mCherry genomic sequences published in GenBank, the deleted viral genomic sequences were input into the sgRNA on-line design website (http// crispr. Mit. Edu), the sgRNA sequences with PAM (NGG) were searched for, and the sgRNA with lower off-target rate was selected and cloned into LENTICRISPR V vector (Addgene, beijing mu gull Biotechnology Co., ltd.) as shown below.

The phosphorylation modification and annealing system of the oligos comprises:

oligo 1(100μM)1μ；

oligo 2(100μM)1μL；

The deionized water is added to 10 mu L;

Treating at 95deg.C for 5min with PCR instrument, and decreasing the temperature to 25deg.C at 5 deg.C per minute

Plasmid enzyme digestion, the reaction system includes:

LENTICRISPR V2 plasmid 2 μg;

BsmBI 2μL；

10X Buffer 2μL

Adding deionized water to 40 mu L;

enzyme cutting at 37 ℃ for 3 hours, running glue and recycling.

The carrier connecting system after the oligo and the enzyme digestion comprises:

oligos dilution of 100 μl;

lentiCRISPR V2 10ng；

10xNEB ligase buffer 1. Mu.L;

NEB ligase 1. Mu.L;

The deionized water is added to 10 mu L;

The mixture is connected for 4 to 6 hours at room temperature, transformation is carried out, cloning is selected, and sequencing is carried out correctly for the next step.

Example 2: construction of the transfer plasmid pMD19T- ΔgI/gE-FCV VP1

The homologous upstream and downstream fragments of the deletion position of gI/gE of FHV Delta gIgE/TK eGFP-mCherry strain and the FCV VP1 expression cassette (SEQ ID NO: 1) are sequentially inserted into a plasmid pMD19T to construct a homologous recombination transport vector pMD 19T-DeltagI/gE-FCV VP1. The amplification primer sequences are shown in Table 1.

TABLE 1 primer sequences related to construction of transfer plasmids

Example 3: construction of the transfer plasmid pMD 19T-delta TK-FPV VP2

The upstream and downstream homologous fragments of the TK deletion position of FHV Delta gIgE/TK eGFP-mCherry strain and an FPV VP2 expression cassette (SEQ ID NO: 2) are sequentially inserted into a plasmid pMD19T to construct a homologous recombination transport vector pMD 19T-DeltagI/gE-FCV VP1. The amplification primer sequences are shown in Table 2.

TABLE 2 primer sequences related to construction of transfer plasmids

Example 4: construction of recombinant viruses

Cas9 plasmid sgRNA-eGFP, the transport vector plasmids pMD 19T-. DELTA.gI/gE-FCV VP1 and pMD 19T-. DELTA.TK-FPV VP2 were co-transfected in CRFK cells for 18 hours using the Lipofectamin 3000 transfection method. After co-transfection, cells were infected with FHV Delta gIgE/TK eGFP-mCherry strain (MOI=0.1). Cells were maintained at 37 ℃ in an incubator with 5% CO ₂, and when 90% CPE occurred, cells were collected and freeze-thawed three times.

Example 5: purification and identification of recombinant viruses

Firstly, CRFK cells are passaged on a six-hole cell plate, and when the cell confluency reaches 90% -100%, the original cell culture solution is discarded and washed for 2 times by PBS; simultaneously, carrying out double dilution on virus stock solution by using EMEM, uniformly inoculating diluted virus on a cell plate by using 10 ^-2-10^-5, incubating for 3 hours in a 37 ℃ incubator, and discarding the virus solution; 2% low melting agarose solution (stored at 60 ℃) was mixed with 2X cell maintenance solution at 1:1, adding into each culture hole at a ratio of 2 ml/hole, cooling, and solidifying to obtain a cover layer. Selecting a fluorescent plaque-free strain through a fluorescence microscope, purifying for 7 rounds to obtain a recombinant FHV delta gIgE/TK FCV VP1-FPV VP2 strain, purifying and preserving the strain in the China center for type culture collection; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 8 and 8 days, and the preservation number is CCTCCNO: V202383.

Example 6: determination of growth characteristics of recombinant viruses

The FHV Delta gIgE/TK FCV VP1-FPV VP2 strain and the FHV Delta gIgE/TK eGFP-mCherry strain are respectively inoculated with CRFK cells which are fully paved with a monolayer according to the MOI=0.1 proportion, and the CRFK cells are placed in an incubator for continuous culture. The virus solution was collected every 6 hours for TCID ₅₀ and a one-step growth curve was drawn (fig. 2). The results showed that the growth curves of FHV Delta gIgE/TK FCV VP1-FPV VP2 and the parent strain FHV Delta gIgE/TK eGFP-mCherry were substantially identical.

Example 7: virus particle electron microscope negative staining observation

Inoculating the strain of the separated FHV delta gIgE/TK FCV VP1-FPV VP2 into CRFK cells, repeatedly freezing and thawing for 3 times after 80% of CPE appears, centrifuging for 30min at 8 000r/min, centrifuging for 60min at 4 ℃ at 12 000r/min, collecting precipitate, performing negative staining with 2% phosphotungstic acid, and observing by an electron microscope. The results show that FCV VP1 and FPV VP2 capsid proteins are able to form virus-like particles in the cytoplasm, and three different sizes of virions can be seen by electron microscopy after superionization of the recombinant virus solution (fig. 3).

Example 8: verification of recombinant viruses

Collecting cell cultures of FHV delta gIgE/TK FCV VP1-FPV VP2 strain and FHV WX19 strain, preparing gel with required concentration by using a product catalog of a manufacturing company, transferring the protein gel after electrophoresis onto an NC film by using a protein semi-dry transfer film instrument, transferring 40 min-60 min under 15V condition, and sealing at 37 ℃ in 5% skim milk for 3h or 4 ℃ overnight. gI, gE, TK, VP1, VP2, gB and Actin antibodies diluted by primary anti-dilution liquid are incubated at room temperature for 4 hours or at 4 ℃ for overnight, then TBST is used for 3 times for 10 minutes, secondary antibodies are incubated at room temperature for 45 minutes, TBST is used for 3 times for 10 minutes, and then a gel development instrument is used for finishing development and storage of photos. The results showed that specific FCV VP1 and FPV VP2 were detected by performing WB, and the results were as expected. (FIG. 4).

Example 9: immunoprotection assay of recombinant viruses

The test method comprises the following steps: selecting 16 cats with herpes virus negative puppets for 3-4 months; randomly dividing into 4 groups, 4 kittens of group A and group B, immunizing 10 ⁶TCID₅₀ recombinant viruses at 0 and nasal drops, and challenged with 10 ⁹TCID₅₀ FCV virulent at 28 days A and C; b and D were challenged with 10 ⁵TCID₅₀ FPV virulent at 28 days. After 14 days of detoxification, body temperature was measured daily and clinical symptoms were observed and recorded.

Test results: as shown in fig. 5-6, all kittens of group C after challenge with virulent FCV exhibited typical clinical symptoms including sneezing, loss of appetite from depression, serous secretion of rhinitis, cough and tongue with canker sore lesions, with later clinical symptoms gradually deepening and death occurring. Group A after the virulent FCV attacks the toxin, the temperature of the kittens is normal, and the two kittens only have the symptom of sneezing on the 4 th day after the toxin attack; all kittens of group D after virulent FPV challenge exhibited typical clinical symptoms including high fever, vomiting, diarrhea, enteritis, and decreased leukocyte numbers, and died. After virulent FPV attacks the virulent, the group B kittens are normal in body temperature, and obvious clinical symptoms of cat fever do not appear. The results show that the FHV delta gIgE/TK FCV VP1-FPV VP2 strain has good protective capability for kittens.

Example 10: preparation of hybridoma cell strain and antibody detection thereof

The hybridoma cell strain is prepared and screened by a conventional test method, and is briefly described as follows:

The purified vaccine strain was used as Freund's complete adjuvant at 1:1 proportion of equal volume emulsification is used as an immune antigen for primary immunization, and 6-8 weeks female BALB/c mice are subcutaneously injected at multiple points, 1 mL/mouse; the same concentration of vaccine strain was used with Freund's incomplete adjuvant at 1:1 proportion of equal volume emulsification for enhancing immunity; the antibody titer was measured by taking blood at day 49 after the first immunization, and BALB/c mice with the highest antibody titer were selected and boosted by intraperitoneal injection with the same concentration of the adjuvant-free vaccine strain 3 days before cell fusion.

The spleen of the immunized mouse is aseptically taken and placed into a cell sieve to prepare spleen cell suspension, and the obtained spleen cells and mouse myeloma cells SP2/0 are mixed according to a ratio of 5:1 was cell fused with a PEG fusion agent, i.e., polyethylene glycol PEG (P7181). The cells were resuspended in HAT medium and sub-packed into 96 well cell culture plates and incubated in a 5% carbon dioxide cell incubator at 37 ℃.

On days 9, 12 and 15 after cell fusion, cell supernatants were taken and positive hybridoma cells were screened using ELISA antibody detection method with vaccine strain as coating antigen. Through square matrix test and various optimization, the concentration of the coating protein is determined to be 10 mug/ml, and the secondary antibody is determined to be 1: 2000. the development time is 10min, a Penton-ELISA detection method is established, and the antibody of the fusion cell supernatant is detected and screened to obtain a positive hybridoma cell strain.

And (3) screening and verifying the screened positive hybridoma cells again by using an ELISA detection method with vaccine strains as coating antigens. Through square matrix test and various condition optimization, the coated virus is determined to be diluted 200 times and the secondary antibody is 1: 2000. the development time is 10min, an ELISA detection method is established, and the hybridoma cell strain which is positive after screening is screened again. Through subcloning screening, a positive Hybridoma cell strain is obtained, the name of the positive Hybridoma cell strain is SHVP, the Hybridoma cell strain SHVP (hybrid oma CELLLINE SHVP) is preserved in China center for type culture collection; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 10 and 11 days, and the preservation number is CCTCC NO: C2023313. the monoclonal antibody secreted by hybridoma cell line SHVP was designated monoclonal antibody VP12.

Specific detection of monoclonal antibodies

Specificity: ELISA test is carried out by taking FHV delta gIgE/TK eGFP-mCherry strain, FHV delta gIgE/TK FCV VP1-FPV VP2 virus strain, FHV-1/WH/2020 strain and FHV-1/WH/2017 strain as coating antigens, and the specificity of monoclonal antibodies in cell culture supernatant of hybridoma cell strain SHVP is detected. The OD450nm value of the sample is larger than 2.1 times that of the negative control is positive (+), the OD450nm value of the sample is smaller than or equal to 2.1 times that of the negative control is negative (-), and the negative control is the cell culture supernatant which is not fused successfully.

The result shows that the monoclonal antibody only specifically binds with FHV delta gIgE/TK FCV VP1-FPV VP2 virus strain and has no cross reaction with other 3 serotype viruses, and the monoclonal antibodies secreted by the hybridoma cell strain of the embodiment and aiming at the vaccine strain related to the application have good specificity.

Stability detection of hybridoma cells

Resuscitates the hybridoma cells in liquid nitrogen at 3 and 6 months, and detects the titer of the antibody in the cell supernatant by ELISA method.

The results show that the ELISA titer is reduced by 2 ¹-2² titer, which shows that the screened monoclonal antibody hybridoma cell strain has good antibody secretion capacity and stability.

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art will appreciate that, in light of the principles of the present invention, improvements and modifications can be made without departing from the scope of the invention.

Claims (8)

1. A recombinant feline herpesvirus genetic engineering vaccine for expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein is characterized in that exogenous feline calicivirus VP1 protein and feline parvovirus VP2 protein are expressed in the recombinant feline herpesvirus genetic engineering vaccine, and gI, gE and TK genes in the feline herpesvirus are deleted.

2. A recombinant feline herpesvirus genetic engineering vaccine expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein according to claim 1, wherein the feline herpesvirus deleted for the gI, gE and TK genes in the feline herpesvirus is the FHV Δ gIgE/TK eGFP-mCherry strain, said FHV Δ gIgE/TK eGFP-mCherry strain, accession No.: v202260, deposited in chinese collection of typical cultures; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2022, 7 and 26.

3. A recombinant feline herpes virus genetically engineered vaccine expressing feline calicivirus VP1 and feline parvovirus VP2 proteins according to claim 1, wherein the genetically engineered vaccine is FHV Δ gIgE/TK FCV VP1-FPV VP2 strain, which FHV Δ gIgE/TK FCV VP1-FPV VP2 strain was deposited in the chinese collection of typical cultures; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 8 and 8 days, and the preservation number is CCTCC NO: V202383.

4. Use of a recombinant feline herpesvirus gene engineering vaccine expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein according to any one of claims 1-3 for the preparation of a medicament for the treatment or prophylaxis of feline herpesvirus type i, feline calicivirus and feline parvovirus.

5. An immune composition characterized by comprising: a recombinant feline herpesvirus vaccine expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein according to any one of claims 1-3; and, a pharmaceutically acceptable carrier.

6. The immune composition of claim 5, wherein: the pharmaceutically acceptable carrier comprises any one or more than two of MONTANIDE ISA206, MONTANIDE ISA201, MONTANIDE GEL 01ST, aluminum hydroxide GEL adjuvant, alumen, freund's adjuvant, lipopolysaccharide, cholesterol, vegetable oil and cytokine; preferably an aluminium hydroxide gel adjuvant.

7. A method for preparing a recombinant feline herpesvirus strain expressing the feline calicivirus VP1 protein and the feline parvovirus VP2 protein according to any one of claims 1 to 3, characterized by comprising the steps of:

(1) Constructing a recombinant vector for expressing cat calicivirus VP1 gene, respectively designing a left recombinant arm and a right recombinant arm of homologous recombination of gI and gE by primer amplification, sequentially assembling a CMV promoter, an FCV VP1 gene fragment and an SV40 polyA signal sequence fragment on the left recombinant arm-CMV-FCV VP1-SV40 polyA-right recombinant arm to obtain a recombinant transfer vector;

(2) Constructing a recombinant vector for expressing cat parvovirus VP2 gene, respectively designing a left recombinant arm and a right recombinant arm for primer amplification TK homologous recombination, a CMV promoter, an FPV VP2 gene fragment and a bGH polyA signal sequence fragment, and sequentially assembling the left recombinant arm-CMV-FPV VP2-bGH polyA-right recombinant arm to obtain a recombinant transfer vector;

(3) The design provides a sgRNA combination for editing eGFP, mCherry comprising the target sequence FHV Δ gIgE/tkegfp-MCHERRY SGRNA, comprising the sequence of SEQ ID NO:3-SEQ ID NO:6, a nucleotide sequence shown in the specification;

(4) After co-transfecting the cells in the step (1) and (2), infecting the cells with FHV delta gIgE/TK eGFP-mCherry strain to obtain the recombinant feline herpesvirus which does not contain eGFP and mCherry fluorescent markers, wherein the recombinant feline herpesvirus is the recombinant feline herpesvirus genetic engineering vaccine FHV delta gIgE/TK FCV VP1-FPV VP2.

8. A recognition antibody for recognizing a recombinant feline herpesvirus genetically engineered vaccine expressing feline calicivirus VP1 protein and feline parvovirus VP2 protein according to any one of claims 1-3, characterized in that said antibody is secreted by Hybridoma cell line SHVP, which Hybridoma cell line SHVP (hybrid mia CELL LINE SHVP 12) is deposited with the chinese collection of typical cultures; the preservation address is: chinese university of Wuhan, post code 430072; the preservation date is: 2023, 10 and 11 days, and the preservation number is CCTCC NO: C2023313.