Celine herpesvirus I type TK gene deletion strain, construction method and application of feline herpesvirus I type TK gene deletion strain

Abstract

The invention discloses a feline herpesvirus type I TK gene deleted strain, a construction method and application thereof, and the feline herpesvirus type I TK gene deleted strain is an FHV-EGFP-delta TK strain obtained by using a feline herpesvirus type I FHV BJ-1 strain as a parent strain and deleting a TK gene through a homologous recombination technology; the deleted TK gene region is replaced by an EGFP (enhanced green fluorescent protein) expression box. According to the TK gene deletion strain, the pathogenicity of the TK gene deletion strain is reduced, and meanwhile good hereditary stability is kept. The invention further provides an FHV-EGFP-delta TK gene deleted live vaccine. The vaccine has the advantages in multiple aspects that mucosal immunity, cellular immunity and humoral immunity are activated at the same time; long-acting immune memory can be induced through transient mucosal infection, and the immunity enhancing frequency is reduced; the production of the vaccine does not need an adjuvant or a complex purification process, and shows good industrial application potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products and relates to a feline herpesvirus type I TK gene deletion strain, a construction method and an application thereof. Background Art

[0002] Feline herpesvirus type 1 (FHV-1) is one of the main pathogens of upper respiratory tract infections and eye diseases in cats. It is highly contagious and pathogenic, and can cause a variety of clinical symptoms such as rhinitis and conjunctivitis. The positive rate in viral upper respiratory tract diseases is as high as 50-75%, and the incidence rate after infection is close to 100%, posing a serious threat to the health of pet cats and wild cats worldwide.

[0003] Although a variety of vaccines are currently available for the prevention of FHV-1 infection, these vaccines only alleviate the clinical symptoms of FHV-1 infection and fail to provide effective immune protection. While traditional inactivated FHV-1 vaccines offer a certain degree of protection, they suffer from short-lived effects and the need for multiple vaccinations, placing an additional financial burden on cat owners. Attenuated vaccines, on the other hand, carry the risk of increased virulence.

[0004] Therefore, the development of a new FHV-1 vaccine has important practical significance. To overcome the above problems, researchers in this field are committed to developing a safer and more effective FHV-1 vaccine. In recent years, gene-deleted vaccines, as an emerging vaccine development strategy, have shown broad application prospects by precisely deleting the key virulence genes of the virus, effectively reducing pathogenicity while retaining its immunogenicity. As a new vaccine strategy, gene-deleted vaccines reduce its pathogenicity while retaining its immunogenicity by deleting the key virulence genes of the virus. Therefore, the development of a vaccine for gene-deleted FHV-1 is of great significance. Summary of the Invention

[0005] The present invention uses feline herpesvirus type 1 FHV BJ-1 strain as the parent strain and utilizes homologous recombination technology to construct a feline herpesvirus type 1 TK gene deleted strain. By knocking out the TK gene, its pathogenicity is reduced while maintaining good genetic stability, providing candidate strains and new ideas for the research and development of relevant drugs and new vaccines for the prevention and treatment of feline herpesvirus infection diseases.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A feline herpesvirus type I TK gene deleted strain is a FHV-EGFP-ΔTK strain obtained by using the feline herpesvirus type I FHV BJ-1 strain as a parent strain and deleting the TK gene through homologous recombination technology; the nucleotide sequence of the TK gene is shown in SEQ ID NO. 1, and the deleted TK gene region is replaced by an EGFP expression cassette.

[0008] The present invention also provides a method for constructing a feline herpesvirus type I TK gene deletion strain, which comprises using homologous recombination technology to construct a feline herpesvirus type I TK gene deletion strain FHV-EGFP-ΔTK: designing primers, amplifying the left and right homologous arms of the TK gene and the EGFP expression cassette, and constructing a transfer vector pUC-EGFP-ΔTK; co-transfecting FHV BJ-1 strain viral genomic DNA and the transfer vector pUC-EGFP-ΔTK into CRFK cells (using a liposome transfection reagent), and obtaining a recombinant virus FHV-EGFP-ΔTK strain through screening.

[0009] The transfer vector pUC-EGFP-ΔTK is constructed by designing primer pairs TK-L and TK-R with reference to the FHV-1 C27 NC-013590 genome; designing primer pairs EGFP box with reference to the full sequence of the pVAX1-EGFP plasmid; using FHV BJ-1 strain genomic DNA as a template, respectively amplifying the left and right homologous arms of the TK gene; amplifying the EGFP expression cassette using EGFP box primers; performing double enzyme digestion on the pUC19 plasmid; subjecting the PCR product and the enzyme digestion product to 1% agarose gel electrophoresis, and recovering and purifying them with an agarose DNA recovery kit after verification; and recovering the TK-L, TK-R and EGFP expression products obtained by gel electrophoresis. box The transfected protein was connected to the linearized pUC19 vector by homologous recombination technology, and the ligation product was transformed into DH5α competent cells for culture and sequencing to obtain the transfer vector pUC-EGFP-ΔTK.

[0010] The specific method for obtaining the recombinant virus FHV-EGFP-ΔTK strain is as follows: the FHV BJ-1 strain genome and the transfer vector pUC-EGFP-ΔTK are co-transfected into CRFK cells, cultured and purified until all the plaques observed under an inverted fluorescence microscope are green fluorescent, and PCR identification is performed using TK check primers to obtain a TK gene-deficient feline herpesvirus type I TK gene-deficient strain.

[0011] The sequences of the primer pair TK-L are shown in SEQ ID NO. 2 and SEQ ID NO. 3; the sequences of the primer pair TK-R are shown in SEQ ID NO. 4 and SEQ ID NO. 5.

[0012] Preferably, the pUC19 plasmid is double-digested using the endonucleases BamHI and HindIII.

[0013] Furthermore, the present invention also provides the use of a TK gene-deficient strain of feline herpesvirus type I in reducing viral virulence, including the use of the TK gene-deficient mutant as a live attenuated vaccine.

[0014] Furthermore, the present invention also provides a feline herpesvirus type I gene-deficient vaccine, the active ingredient of which comprises the feline herpesvirus type I TK gene-deficient strain or a culture thereof.

[0015] The single immunization dose of the vaccine is ≥10 5 TCID 50 / dose.

[0016] Preferably, the vaccine is suitable for use in felines.

[0017] Beneficial effects of the present invention:

[0018] First, using a pUC19-based transfer vector system, precise deletion of the TK gene and insertion of the EGFP reporter gene were achieved through double enzyme digestion and homologous recombination. The results showed that the approximately 3590 bp fragment amplified by the M13 primer verified the correct construction of the transfer vector pUC-EGFP-ΔTK, and sequencing results fully matched the expected structure, demonstrating the rationality of the homology arm design (TK-L / TK-R). Furthermore, after co-transfection, a recombinant strain stably expressing EGFP was obtained after only 10 rounds of plaque purification, demonstrating the high efficiency of homologous recombination using this method.

[0019] Secondly, the use of EGFP as a marker gene in the FHV-1 gene-deleted strain not only provides an efficient screening tool for plaque purification but also enables clearer differentiation between recombinant virus (green fluorescence) and wild-type virus (non-fluorescent). Furthermore, the sustained expression of EGFP (stable after 10 generations of purification) indicates its successful integration into non-essential regions of the viral genome without interfering with key gene functions. This not only provides a visualization tool for tracking viral dynamics but also serves as a molecular marker for distinguishing vaccine strains from wild-type strains, meeting biosafety regulatory requirements.

[0020] Again, in terms of genetic stability, FHV-EGFP-ΔTK still stably expressed green fluorescence after 20 consecutive passages, and PCR verification confirmed that no reversion mutation occurred in the TK gene deletion region, indicating that the homologous recombination combined with exogenous gene insertion strategy is feasible in this study.

[0021] Finally, TK gene deletion is a key factor in terms of safety and potential application. Studies have shown that TK, a core factor in herpesvirus replication and latent infection in neurons, effectively inhibits viral migration to the trigeminal ganglion, significantly reducing the risk of latent infection. Furthermore, TK deletion weakens the virus's inhibitory effect on host interferon pathways (such as STAT1 signaling), significantly enhancing the innate immune response, thereby limiting viral spread and effectively reducing virulence. Compared to commercially available inactivated vaccines, the FHV-EGFP-ΔTK gene-deficient live vaccine offers several advantages. In terms of immunogenicity, the live vaccine simultaneously activates mucosal, cellular, and humoral immunity, while the inactivated vaccine primarily relies on antibody responses. Regarding the duration of immune protection, the TK-deficient strain can induce long-lasting immune memory through a brief mucosal infection, reducing the frequency of booster immunizations. From a production cost perspective, this live vaccine requires no adjuvants or complex purification processes, and the single immunization dose is only 10⁵ TCID⁵ / dose, demonstrating excellent potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the gel electrophoresis of PCR with universal primer M13 for the transfer vector pUC-EGFP-ΔTK, where M: DL5000 Marker; 1: single positive clone;

[0023] Figure 2 This is the plasmid map of the transfer vector pUC-EGFP-ΔTK;

[0024] Figure 3 Electron micrograph of CRFK cells co-transfected with pUC-EGFP-ΔTK and FHV-1 BJ-1 for 72 h (100×);

[0025] Figure 4 Electron micrographs of FHV-EGFP-ΔTK purification (100×), where F1: purification result of the first generation; F5: purification result of the fifth generation; F10: purification result of the tenth generation;

[0026] Figure 5 This is the gel electrophoresis of PCR for FHV-EGFP-ΔTK using TK check primers, where M: DL 2000Marker; 1: TK check;

[0027] Figure 6 Electron micrographs of FHV-EGFP-ΔTK (100×), where A, B, and C represent the 1st, 10th, and 20th generations of FHV-EGFP-ΔTK;

[0028] Figure 7PCR gel electrophoresis of FHV-EGFP-ΔTK strain; M: DL2000 DNA Marker; 1-5: FHV-EGFP-ΔTK generation 1, 5, 10, 15, and 20;

[0029] Figure 8 Growth curves of FHV BJ-1 and FHV-EGFP-ΔTK;

[0030] Figure 9 The following diagrams show the clinical symptoms of the three groups of test cats on the second, fourth, and sixth days after infection. A, B, and C are the first, second, and third groups of test cats, respectively.

[0031] Figure 10 The following are statistical graphs of body temperature, clinical scores, virus shedding and survival rates of the three groups of experimental cats after infection. DETAILED DESCRIPTION

[0032] In order to explain the present invention more clearly, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0033] Example 1

[0034] 1 Test materials

[0035] 1.1 Strains, plasmids, and cells:

[0036] The feline herpesvirus type 1 (FHV) BJ-1 strain used in this application was isolated from nasal swabs and lungs of a cat with respiratory symptoms. It is described in the literature (Tao Weijie, Liu Xuejiao, Song Xiaoli, Cheng Baoyu, Yu Yongle, Shan Hu, Zhang Chuanmei, "Isolation, Identification, and Biological Characterization of Feline Herpesvirus Type 1," Acta Virologica Sinica, Vol. 39, No. 4, pp. 1053-1061) and maintained in the applicant's laboratory. The pUC19 plasmid was purchased from Beijing Qingke Biotechnology Co., Ltd.; the pVAX1-EGFP plasmid was a gift from Professor Li Junwei of Qingdao Agricultural University; and CRFK cells were purchased from Shanghai Yubo Biotechnology Co., Ltd.

[0037] 1.2 Main Reagents

[0038] Restriction enzymes QuickCut™ BamHI and HindIII, and loading buffer were purchased from Bioray Biotechnology (Beijing) Co., Ltd. Fetal bovine serum (FBS), endotoxin-free plasmid extraction kit, 2×T5 SuperPCR Mix, 2×CE Mix, Lip2000 Transfection Reagent, and DNA marker were purchased from Nanjing Novozymes Biotechnology Co., Ltd. Opti-MEM medium, Dulbecco's Modified Eagle ...

[0039] 1.3 Experimental Animals

[0040] Nine 3-month-old rural cats were tested for feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), feline parvovirus (FPV), and feline coronavirus (FCoV) antigens using an animal-specific immunofluorescence quantitative analyzer (Haiweite (Guangzhou) Medical Technology Co., Ltd.), and the results were negative for FHV-1 antibodies.

[0041] 2 Primer design

[0042] Primers for amplifying the homology arms and the green fluorescent expression cassette were designed using CE Design V1.04 software, based on the FHV-1 C27 (NC-013590) genome sequence and the complete pVAX1-EGFP plasmid sequence accessed from GenBank (https: / / www.ncbi.nlm.nih.gov / ). The TK gene deletion detection primer, TK check, and the M13 universal primer, M13, were designed based on the predicted TK gene deletion and recombinant transfer vector detection (Table 1). These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the TK gene is shown in SEQ ID NO. 1.

[0043] Table 1 Primer sequence information

[0044]

[0045] 3. Construction of transfer vector

[0046] Using the FHV BJ-1 genome as a template, primers TK-L and TK-R were used to amplify the left and right homology arms of the TK gene, respectively. Primers for the EGFP box were used to amplify the EGFP expression cassette. The pUC19 plasmid was double-digested with QuickCut™ BamHI and QuickCut™ HindIII. The PCR and digestion products were verified by electrophoresis on a 1% agarose gel and purified using an agarose DNA purification kit.

[0047] The TK-L, TK-R, and EGFP expression cassettes recovered from the gel were ligated into the linearized pUC19 vector by homologous recombination. The ligation products were transformed into DH5α competent cells and cultured in an inverted manner at 37°C on LB solid medium containing 100 μg / mL ampicillin for 16 h. A single positive clone was picked and verified by PCR using the M13 universal primer. After sequencing, the correctly ligated transfer vector was named pUC-EGFP-ΔTK.

[0048] The gene sequences of TK-L, TK-R and EGFP expression cassettes obtained by PCR are shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively.

[0049] The pUC-EGFP-ΔTK was verified by PCR using the M13 universal primer. The product was electrophoresed on a 1% agarose gel and observed using an automatic gel imaging system. The results are shown in Figure 2. Figure 1 The target band size is about 3590 bp, and the gene sequence is shown in SEQ ID NO. 15. The sequencing results are consistent with the prediction, proving that the transfer vector pUC-EGFP-ΔTK was successfully constructed. The transfer vector map is shown in Figure 2 shown.

[0050] 4 Construction and identification of recombinant virus FHV-EGFP-ΔTK

[0051] FHV BJ-1 strain genomic DNA and pUC-EGFP-ΔTK were co-transfected into well-growing CRFK cells using Lipomaster 2000 transfection reagent. The culture medium was changed 12 h after transfection, and the cells were cultured until the cytopathic effect reached 80%. The virus solution was harvested by freezing and thawing three times and plaque purification was performed until all plaques observed under an inverted fluorescence microscope were green fluorescent. FHV-EGFP-ΔTK was identified by PCR using TKcheck primers.

[0052] After 72 h of co-transfection of CRFK cells with pUC-EGFP-ΔTK and FHV BJ-1 (MOI = 0.1), the CPE of the cells reached 80%. Figure 3After freezing and thawing three times, the cells were centrifuged and the supernatant was filtered through a 0.22 μm bacterial filter for plaque purification.

[0053] The harvested virus solution was diluted in 10-fold gradients, and 10 -2 ~10 -6 For plaque purification, pick green fluorescent plaques under an inverted fluorescence microscope and add them to 1 mL of DMEM medium. After 10 rounds of plaque purification, all plaques emit green fluorescence. Figure 4 The genomic DNA of the virus was extracted after 10 rounds of plaque purification and tested by PCR using TK check primers. The amplified product was electrophoresed on 1% agarose gel to obtain a 1750 bp target fragment. Figure 5 The product was sequenced and was consistent with the prediction, and a TK gene-deficient feline herpesvirus type Ⅰ TK gene-deficient strain was obtained.

[0054] 5. Determination of genetic stability of FHV-EGFP-ΔTK

[0055] FHV-EGFP-ΔTK was continuously propagated on CRFK cells to the 20th generation, and the TCID of the 1st, 5th, 10th, 15th and 20th generations were determined by the Reed-Meunch method. 50 , and extracted viral supernatant DNA, and performed PCR and sequencing identification using TK check primers.

[0056] The FHV-EGFP-ΔTK strain was continuously passaged in the CRFK cell line to the F20 generation, and the CPE and fluorescence expression of each generation were observed using an inverted fluorescence microscope. Figure 6 All passages of the virus induced typical CPE, and cells induced by the virus at each passage emitted green fluorescence. This demonstrates that FHV-EGFP-ΔTK maintained stable fluorescent marker expression and viral replication during serial passages.

[0057] DNA was extracted from the viral supernatants of the FHV-EGFP-ΔTK strain F1, F5, F10, F15, and F20 passages, and specific PCR detection was performed using primer TK-check, and the results were all positive ( Figure 7 ).

[0058] The virus titers of FHV-EGFP-ΔTK F1, F5, F10, F15, and F20 generations of virus solution were measured. The titer of F1 generation virus was 10 5.70 TCID 50 / 0.1 mL, the F5 virus titer is 10 5.67 TCID 50 / 0.1 mL, the F10 virus titer is 10 5.80TCID 50 / 0.1 mL, the F15 virus titer is 10 5.50 TCID 50 / 0.1 mL, the F20 virus titer is 10 5.62 TCID 50 / 0.1 mL. The virus titer of each generation was 10 5.50 ~10 5.80 TCID 50 The titer of the FHV-EGFP-ΔTK strain fluctuated within the range of 0.1 mL / min, indicating that the virus titer of the FHV-EGFP-ΔTK strain maintained a high stability during serial passage.

[0059] 6 One-step growth curve drawing of FHV-EGFP-ΔTK

[0060] FHV-EGFP-ΔTK and FHV BJ-1 were infected into CRFK cells grown in monolayers (MOI = 0.1), with three replicates in each group. Virus fluids were harvested at 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, and 72 h after infection, and the virus titers were determined. The TCID was calculated using the Redd-Muench method. 50 Graphpad Prism 10.1 software was used to draw the one-step growth curve.

[0061] To further clarify the biological characteristics of FHV-EGFP-ΔTK, the virus titer was determined by collecting virus cell culture fluid at different time points and plotting the one-step growth curve of FHV-EGFP-ΔTK to determine its growth characteristics. The results showed that the growth curve of the TK gene deletion strain was similar to that of the parent strain, but the virus titer was slightly reduced ( Figure 8 ).

[0062] 7 Safety Tests

[0063] The harvested FHV-EGFP-ΔTK and FHV BJ-1 virus solutions were adjusted to a viral titer of 1×10 6 TCID 50 Nine 3-month-old kittens were randomly divided into three groups. Groups 1, 2, and 3 were infected with 1 mL of FHV BJ-1, 1 mL of FHV-EGFP-ΔTK, and 1 mL of DMEM, respectively. Clinical signs of the cats were observed daily and scored according to the standard FHV-1 clinical score established by the United States Department of Agriculture (USDA).

[0064] The results are as follows Figure 9 、 Figure 10 As shown:

[0065] Two days after reinfection with FHV BJ-1, cats in Group 1 began to show initial symptoms, including sneezing and watery eyes, accompanied by a fever exceeding 39.5°C. Four days later, they developed significant mucous discharge from their eyes and nose, with their eyes closing more than 75% of the time. They also developed severe conjunctivitis, nasal congestion, sneezing, and a severe decrease in spirits and appetite. Six days after infection, symptoms worsened, with the infected cats experiencing increased eye redness and swelling, completely closing their eyes and becoming unable to open. Nasal obstruction led to mouth breathing. Two cats developed cyanosis and dehydration, and died on days 7 and 10 after immunization, respectively.

[0066] The cats in group 2 showed milder symptoms after infection with FHV-EGFP-ΔTK, with only mild symptoms such as runny nose and sneezing appearing 2 to 6 days after infection. There was less eye and nasal secretions, no severe conjunctivitis or difficulty breathing, and their spirits and appetite were basically normal. There was no cyanosis on the skin. Their overall health was significantly better than that of the FHV BJ-1 group, and all the cats survived.

[0067] The cats in the blank control group of group 3 were in good condition throughout the experiment, without fever or other clinical symptoms.

[0068] The experimental results showed that compared with the FHV B-1 strain, the clinical symptoms after FHV-EGFP-ΔTK infection were milder, the experimental cats could recover on their own 6 days after infection, and there was no risk of death. Its safety was much higher than the parent strain FHV BJ-1.

Claims

1. A feline herpesvirus type I TK gene deletion strain, characterized in that: The deletion strain is an FHV-EGFP-ΔTK strain obtained by using the feline herpesvirus type I BJ-1 strain as the parent strain and deleting the TK gene through homologous recombination technology; the nucleotide sequence of the TK gene is shown in SEQ ID NO. 1, and the deleted TK gene region is replaced by the EGFP expression box.

2. The method for constructing the feline herpesvirus type Ⅰ TK gene deletion strain according to claim 1, characterized in that: The method comprises the following steps: utilizing homologous recombination technology to construct a feline herpesvirus type I TK gene deletion strain FHV-EGFP-ΔTK; designing primers, amplifying the left and right homologous arms of the TK gene and an EGFP expression box, and constructing a transfer vector pUC-EGFP-ΔTK; and co-transfecting FHV BJ-1 strain viral genomic DNA and the transfer vector pUC-EGFP-ΔTK into CRFK cells, and obtaining a recombinant virus FHV-EGFP-ΔTK strain through screening.

3. The construction method according to claim 2, wherein: The transfer vector pUC-EGFP-ΔTK is constructed by designing primer pairs TK-L and TK-R with reference to the FHV-1 C27 NC-013590 genome; designing primer pairs for the EGFP expression cassette with reference to the full sequence of the pVAX1-EGFP plasmid; using the FHV BJ-1 strain genome as a template, amplifying the left and right homologous arms of the TK gene, respectively; amplifying the EGFP expression cassette using EGFP expression cassette primers; performing double enzyme digestion on the pUC19 plasmid; subjecting the PCR product and the enzyme digestion product to 1% agarose gel electrophoresis, and recovering and purifying them using an agarose DNA recovery kit after verification; ligating the TK-L, TK-R, and EGFP expression cassettes recovered from the gel to the linearized pUC19 vector through homologous recombination technology, and transforming the ligation products into DH5α competent cells for culture and sequencing to obtain the transfer vector pUC-EGFP-ΔTK.

4. The construction method according to claim 2, wherein: The specific method for obtaining the recombinant virus FHV-EGFP-ΔTK strain is as follows: the FHV BJ-1 strain genome and the transfer vector pUC-EGFP-ΔTK are co-transfected into CRFK cells, cultured and purified until all the plaques observed under an inverted fluorescence microscope are green fluorescent, and PCR identification is performed using TK check primers to obtain a TK gene-deficient feline herpesvirus type I TK gene-deficient strain.

5. The construction method according to claim 3, wherein: The sequences of the primer pair TK-L are shown in SEQ ID NO. 2 and SEQ ID NO. 3; the sequences of the primer pair TK-R are shown in SEQ ID NO. 4 and SEQ ID NO.

5.

6. The construction method according to claim 3, wherein: The pUC19 plasmid was double-digested with BamHI and HindIII.

7. Use of the feline herpesvirus type Ⅰ TK gene deleted strain according to claim 1 in reducing viral virulence.

8. The use according to claim 7, characterized in that: The application includes the application of the mutant strain with TK gene deletion as a live attenuated vaccine.

9. A feline herpesvirus type 1 gene-deleted vaccine, characterized in that: The active ingredient comprises the feline herpesvirus type I TK gene deleted strain or its culture as claimed in claim 1.

10. The vaccine according to claim 9, wherein The single immunization dose of the vaccine is ≥10 5 TCID 50 / dose.

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