Duck plague virus UL51 gene deleted strain as well as construction method and application thereof

Abstract

The invention discloses a duck plague virus UL51 gene deletion strain and a construction method and application thereof, the deletion strain is preserved in China Center for Type Culture Collection in Wuhan University in China on April 2, 2025, and the preservation number is CCTCC NO: V202523. The method comprises the following steps: deleting a UL51 gene to obtain an infectious clone pDPV CHv-deltaUL51 of a UL51 gene deleted strain, and transfecting a duck source cell with the infectious clone DNA to obtain the UL51 gene deleted virus strain DPV CHv-deltaUL51. Detecting immune ducklings infected by viruses, and finding that the deletion of the UL51 gene weakens the duplication of DPV in the ducks and reduces the pathogenicity of the ducks; after the immunized duck is attacked by the DPV virulent CHv strain, the DPV virulent CHv strain can be prevented from lethal attack. The virus strain constructed by the invention has reduced pathogenicity to ducks, and has the potential of being used as a universal vector of an attenuated vaccine or a duck live vector vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, and particularly relates to a duck plague virus UL51 gene deletion strain and a construction method and application thereof. BACKGROUND

[0002] Duck plague virus (DPV), also known as duck enteritis virus (DEV), is the pathogen causing acute and septicemic infectious disease of ducks, geese, swans and other anseriform birds. The sick ducks have swollen head and neck, high temperature, increased secretion of eyelid and cloaca, and reduced feeding and activity. The autopsy shows that the immune organs and internal organs are hemorrhagic and the blood vessels are damaged. The virus mainly proliferates in the mucosa of the digestive tract, and can proliferate widely in the host's organs such as liver, spleen, intestinal tract and bursa of Fabricius. Due to its fast transmission speed, wide transmission range, high morbidity and mortality, it has become one of the main diseases that seriously endanger the production of waterfowl. Analyzing the pathogenesis of DPV can take more effective comprehensive prevention and control measures; and vaccine immunization is one of the most effective measures to prevent DPV infection. The current vaccine is the traditional inactivated vaccine and the attenuated vaccine, the former has high production cost, and the use of adjuvant may cause harm to the immunized animals, and even residues in animal products may affect human health; the latter is a natural method to select and screen strains, and the genetic background of the strain is unclear, and the potential consequences of genetic mutation cannot be predicted. In order to control the harm of DPV, it is necessary to expound the pathogenesis of DPV and develop a new generation of vaccine with low cost, safety and effectiveness.

[0003] DPV belongs to the subfamily of alpha herpes virus, the genus of Marek's disease virus ( Mardivirus ), and the virus particle is spherical with a diameter of 160-180 nm, which is composed of linear double-stranded DNA genome, capsid, cortex and envelope from inside to outside. The protein layer between the capsid and the envelope is the cortex, which is a unique structure of herpes virus and has multiple functions. Understanding the function of the cortex protein encoded by DPV in virus replication and pathogenesis can provide ideas for effective control of duck plague. The cortex protein encoded by the UL51 gene of other herpes viruses plays a wide role in the life cycle of the virus, affecting the replication of the virus and the primary and secondary envelope coating, regulating the assembly of the virus and the transmission of the virus between cells. Through analysis of the genome and UL51 gene (genbank accession number NO. JQ647509.1) of DPV-CHv, it is found that the UL51 gene is 759 bp in length, encoding a 33 kDa cortex protein containing 4 potential phosphorylation sites and 1 acylation site, and also containing a Golgi localization signal, indicating that it may be located in the Golgi body in the cytoplasm. However, the biological function of DPV UL51 protein in virus replication and infection is still unclear.

[0004] Gene deletion is the basis for studying the function of viral genes. The earliest gene deletion of DPV is to construct a transfer vector containing the homologous sequences (generally about 1 kb) on both sides of the target gene, and to obtain a deletion virus by homologous recombination in cells after transfection. The molecularly cloned virus based on bacterial artificial chromosome (BAC) recombines the whole genome of DPV into BAC to construct a BAC recombination DPV rescue system. Meanwhile, the Red / ET modification technology is combined to complete the gene deletion and exogenous gene insertion of DPV in the prokaryotic system by using mature gene manipulation means. However, after the gene deletion of DPV on the platform of BAC recombination DPV rescue system by using the Red / ET modification technology, two FRT sites and MiniF elements are left at the deletion site. The residual FRT exogenous site and MiniF elements have an impact on the exploration of gene function, the development of live attenuated vaccine and the license. The BAC platform DPV CHv-BAC-G developed in recent years solves the problem of residual MiniF elements, but the deletion virus obtained by the first targeting can proliferate in vitro cells and cannot infect ducks, so the pathogenicity evaluation cannot be performed. The virus obtained by the second targeting can infect ducks for pathogenicity evaluation, and the process is complicated and the workload is large. SUMMARY

[0005] The purpose of the present application is to provide a duck plague virus UL51 gene non-scarring deletion strain DPV CHv-ΔUL51 and a construction method thereof, which can effectively solve the problem of residual bases and MiniF elements at the deletion site when deleting the DPV gene, and the operation is simple, only one targeting is needed, and the deletion virus obtained by rescue can be evaluated for pathogenicity. The constructed duck plague virus UL51 gene deletion strain DPV CHv-ΔUL51 has reduced virulence, good genetic stability, and can resist 100 LD 50 of duck plague virulent virus attack, and has the potential as a gene deletion vaccine. At the same time, since the DPV CHv-ΔUL51 has the potential as a vaccine, the infectious clone pDPV CHv-ΔUL51 can be used as the basis for preparing duck live vector vaccine and duck plague and other diseases dual / multivalent vaccine, and for preparing live vector vaccine and dual or multivalent live vaccine.

[0006] In order to achieve the above purpose, the present application provides a construction method and application of duck plague virus UL51 gene non-scarring deletion strain DPV CHv-ΔUL51.

[0007] Specifically, the present application provides a duck plague virus UL51 gene deletion strain DPV CHv-ΔUL51 ( Mardivirus ), which has been preserved in the China Center for Type Culture Collection of Wuhan University on April 2, 2025, and the preservation number is CCTCC NO: V202523.

[0008] The gene deletion strain provided by the application can be used in the research on the mechanism of the UL51 gene and the protein coded by the UL51 gene affecting the pathogenicity of duck plague virus.

[0009] The gene deletion strain provided by the application can be used in the preparation of attenuated vaccine of duck plague virus.

[0010] The gene deletion strain provided by the application can also be used in the field of duck breeding. The UL51 gene deletion strain can be used to prepare live vector vaccine or double / multiple attenuated vaccine of other diseases, and can be used in the prevention and control of infectious diseases caused by duck plague virus and other pathogenic infections.

[0011] The application further provides a construction method of the duck plague virus UL51 gene deletion strain. (1) obtaining a targeting fragment The UL51 gene deletion targeting fragment is obtained by using the pEP-Kan-S plasmid as a template and performing PCR amplification with primers with nucleotide sequences shown in SEQ ID NO. 1-2. (2) first homologous recombination to delete the target gene The obtained UL51 gene deletion targeting fragment is added to the competent bacteria, mixed, and then subjected to electroporation and plating. Single colonies are picked and used as templates for single colony PCR identification with primers with nucleotide sequences shown in SEQ ID NO. 7-8. Positive clone bacteria in which the Kan resistance gene replaces the UL51 gene are screened. (3) second homologous recombination to delete the Kan resistance gene The positive clone bacteria obtained in step (2) are inoculated into LB / Cm, and cultured at 30°C and 180 r / min overnight to obtain a seed liquid. The seed liquid is added to LB / Cm, and cultured at 30°C and 180 r / min. L-arabinose is added, and the bacteria are cultured at 30°C and 220 r / min. The bacteria are cultured at 42°C and 180 r / min, and then cultured at 30°C and 180 r / min. The bacteria liquid is mixed with LB, and then plated on LB / Cm plates and cultured at 30°C. Several single colonies are picked and plated on LB / Cm+Kan and LB / Cm plates, respectively, and cultured at 30°C. Colonies that do not grow on the LB / Cm+Kan plate but grow on the LB / Cm plate are selected for PCR identification with primers with nucleotide sequences shown in SEQ ID NO. 7-8. The correct bacteria are the clone bacteria with the duck plague virus UL51 gene deletion strain infectious clone pDPV CHv-ΔUL51. (4) virus rescue The pDPV CHv-ΔUL51 plasmid DNA is extracted and transfected into DEF cells. When cytopathic effect occurs, the culture sample is collected and inoculated into fresh DEF cells to obtain the UL51 gene deletion virus DPV CHv-ΔUL51.

[0012] Preferably, in step (1) of the above construction method, The PCR amplification system is: PrimeSTAR Max DNA Polymerase, primers ΔUL51-F / R with nucleotide sequences shown in SEQ ID NO. 1~2, pEP-Kan-S plasmid and ddH2O; The PCR amplification procedure is: 98℃, 2min; 98℃, 10s, 55℃, 15s, 72℃, 5s, for a total of 34 cycles; 72℃, 10min; 16℃, ∞.

[0013] Preferably, in step (2) of the above construction method, the competent bacteria are GS1783-pDPV-CHv50; and the preparation method is: streaking the GS1783-pDPV-CHv50 strain on an LB / Cm plate, culturing overnight at 30℃; picking a single colony to inoculate in LB / Cm, culturing overnight at 30℃, 180r / min as a seed liquid; inoculating the seed liquid in LB / Cm, culturing at 30℃, 220r / min until the OD is 0.5, then transferring to 42℃, 220r / min, and immediately placing in an ice bath for standing; centrifuging at 4℃ to remove the supernatant, blowing the bacterial body with 4℃ pre-cooled sterilized ddH2O, centrifuging at 4℃ to remove the supernatant, adding ddH2O, and obtaining the competent bacteria.

[0014] Preferably, in step (2) of the above construction method, the single colony PCR identification system is: 2×Hieff Robust PCR Master Mix, a single colony, primers UL51-JD-F / R with nucleotide sequences shown in SEQ ID NO. 7~8, and ddH2O; and the PCR identification procedure is: 94℃, 5min; 94℃, 30s, 55℃, 30s, 72℃, 90s, for a total of 34 cycles; 72℃, 7min; 16℃, ∞.

[0015] The present application has the following advantages: In order to obtain a duck plague virus gene deletion strain without foreign base residues, the present application is based on a bacterial artificial chromosome recombination DPV rescue system platform (GS1783-pDPV-CHv50 strain), and uses a Red-based modification technology, i.e., using a GS1783 E. coli strain containing a Red operon and I_ Sce I enzyme gene sequence and a pEP-Kan-S plasmid containing a Kanamycin (Kan) resistance gene and I_ SceThe UL51 gene of the duck plague virus is deleted by replacing the UL51 gene with a Kan resistance gene through homologous recombination on a bacterial artificial chromosome recombination DPV rescue system platform, and then removing the Kan resistance gene through secondary homologous recombination, so that an infectious clone pDPV CHv-ΔUL51 of the duck plague virus UL51 gene deletion strain is obtained; and the pDPV CHv-ΔUL51 is transfected into a duck-derived cell to complete construction of the scarless duck plague virus UL51 gene deletion strain DPV CHv-ΔUL51.

[0016] The application first constructs a scarless deletion strain of the duck plague virus UL51 gene, and the virus of the constructed deletion strain has significantly reduced virulence and can resist 100 LD 50 of the DPV virulent virus, and has the potential to be used as a universal carrier for preparing a DPV attenuated deletion live vaccine and a duck live vector vaccine. The application also has the potential to be used as a basis for preparing a two or multi-vaccine for duck plague and other diseases, and has the potential to be used for preparing a two or multi-live vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A schematic diagram for construction of the infectious clone of the duck plague virus UL51 gene deletion and the revert strain in Example 1 of the application, wherein A is GS1783-pDPV CHv-ΔUL51; and B is GS1783-pDPV CHv-ΔUL51Rev.

[0018] Figure 2 A plasmid map of pEP-Kan-S in Example 1 of the application.

[0019] Figure 3 An RFLP identification diagram of the infectious clone of the duck plague virus UL51 gene deletion strain in Example 2 of the application.

[0020] Figure 4 Rescue results of DPV CHv-ΔUL51 in Example 3 of the application.

[0021] Figure 5 PCR electrophoresis identification (A) of DPV CHv-ΔUL51 in Example 4 of the application and Western blot identification (B) of DPV CHv-ΔUL51 in Example 5 of the application.

[0022] Figure 6 A multi-step growth curve of DPV CHv-ΔUL51 in Example 5 of the application, wherein A is a virus TCID 50 B is a virus genome copy number analysis, and data analysis is performed by T test, and the significance is marked as ns: no significant difference; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0023] Figure 7 A is the experimental flow chart of virus infection of duckling; B, C and D are rectal temperature change (B), body weight change (C) and survival (D) of infected duck, respectively.

[0024] Figure 8 A is the experimental flow chart of virus infection of duckling; B, C and D are rectal temperature change (B), body weight change (C) and survival (D) of infected duck, respectively.

[0025] Figure 9 A is the experimental flow chart of virus infection of duckling; B, C and D are rectal temperature change (B), body weight change (C) and survival (D) of infected duck, respectively.

[0026] Figure 10 A is the experimental flow chart of virus infection of duckling; B, C and D are rectal temperature change (B), body weight change (C) and survival (D) of infected duck, respectively.

[0027] Figure 11 A is the experimental flow chart of virus infection of duckling; B, C and D are rectal temperature change (B), body weight change (C) and survival (D) of infected duck, respectively. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0029] It should be noted that, unless otherwise specified, the experimental methods in the following examples are all conventional methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents and the like used in the following examples are commercially available, unless otherwise specified.

[0030] Duck plague virus China virulent strain (DPV-CHv) (genbank accession number: NO. JQ647509.1) was isolated, preserved and provided by Cheng Anchun et al. of Guizhou University Animal Science and Technology College, and has an LD50 of 10-3.5 for 28-day-old ducklings. 50 is 10 8 / 1mL; The GS1783-pDPV-CHv50 strain (DPV-CHv was uploaded to duck embryo fibroblasts (DEF) for 50 generations, and the viral genomic DNA was extracted, inserted into the bacterial artificial chromosome plasmid, and then transformed into GS1783 Escherichia coli competent cells to obtain an infectious clone containing the complete DPV genome sequence) was constructed, preserved and provided by Cheng Anchun et al. of the College of Animal Science and Technology of Guizhou University; pEP-Kan-S plasmid was preserved and provided by Cheng Anchun et al. of the College of Animal Science and Technology, Guizhou University; duck plague commercial vaccine: CVCC-AV1222, purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. (Veterinary Drug Production Permit (2016) 080072024).

[0031] The experimental animals used in the following experiments were 14-day-old ducklings and 9-11-day-old duck embryos, which tested negative for DPV and DPV antibodies.

[0032] Example 1: Construction of DPV-UL51 gene deletion strain 1. Construction principle of duck plague virus gene deletion strain The construction principle of duck plague virus deletion strains (taking DPV CHv-ΔUL51 as an example) can be found in [link to documentation]. Figure 1 As shown in A in Table 1, the primers used to construct the deletion strain are ΔUL51-F / R, as shown in SEQ ID NO. 1 and 2. Based on the existing DPV bacterial artificial chromosome platform in the laboratory (GS1783-pDPV-CHv50 strain), the UL51 gene was deleted without scarring using Red homologous recombination technology to obtain DPVCHv-ΔUL51. For the reversion virus DPV CHv-ΔUL51 Rev (which restores UL51 to DPV CHv-ΔUL51), see [link to documentation]. Figure 1 As shown in B in Table 1, its construction principle and process are basically the same as those of DPV CHv-ΔUL51. The primers used to construct the revertant strain are shown in Table 1 as UL51-Rev-F / R and UL51-Rev-Kan-F / R, which are SEQ ID NO.3~4 and SEQ ID NO.5~6.

[0033] Table 1 Primers

[0034] 2. Knockout of the UL51 gene (1) Obtain shooting footage pEP-Kan-S bacteria (see...) Figure 2 (As shown in the figure) Incubate overnight at 37°C on LB / Kan plates, pick a single colony and inoculate it into 100 mL LB, incubate overnight at 37°C and 180 r / min; extract pEP-Kan-S plasmid as template, and use ΔUL51-F / R as primers for PCR amplification of the target fragment.

[0035] Take the deletion of UL51 targeting fragment as an example, its amplification system is: 5 μL PrimeSTAR ® Max DNA Polymerase, 0.2 μL of ΔUL51-F / R primer, 0.2 μL of pEP-Kan-S and 4.4 μL of ddH2O. The amplification procedure is: 98℃, 2 min; 98℃ 10 s, 55℃ 15 s, 72℃ 5 s, cycle 34 times; 72℃, 10 min; 16℃, ∞.

[0036] The electrophoresis result of PCR amplification product shows that the size of targeting fragment (Kan resistance gene fragment, see Figure 1 ‘B+a+Kan+A’ fragment in FIG. 1) is consistent with the theoretical value 1051 bp, indicating that the amplified targeting fragment is correct.

[0037] (2) First homologous recombination to delete the target gene After the GS1783pDPV-CHv50 bacteria were cultured on LB / Cm (chloramphenicol) plate at 30℃ overnight, they were inoculated into 5 mL LB / Cm, and cultured at 30℃, 180 r / min overnight as seed liquid; the seed liquid was inoculated into 50 mL LB / Cm, and cultured at 30℃, 220 r / min until OD 490 was about 0.5; 42℃, 220 r / min for 15 min, immediately placed in ice bath for 20 min; 4℃, 4700 r / min centrifugation for 10 min to remove supernatant; 4℃ pre-cooled 40 mL sterilized ddH2O was repeatedly inverted to clean the bacterial body; 4℃, 4700 r / min centrifugation for 10 min to remove supernatant; repeated 5 times. Add ddH2O to constant volume to 500 μL, which is the prepared competent bacteria, ready for use.

[0038] 200 ng of UL51 gene deletion targeting fragment was added to the competent bacteria and mixed, and then electroporation was performed at 2.5 kv; 800 μL of LB was added, and the bacteria were cultured at 30℃, 220 r / min for 1 h; 4500 r / min centrifugation for 5 min to remove supernatant, 150 μL of LB was added to scatter the bacterial body, and then uniformly coated on LB / Cm+Kan plate, and cultured at 30℃ for 48 h.

[0039] Single colonies were streaked on LB / Cm+Kan plates and incubated at 30°C for 24 h; single colonies were picked and placed in prepared PCR reaction solution, and UL51 deletion bacteria were identified by PCR using primers UL51-JD-F / R (SEQ ID NO. 7~8) in Table 1 and sequenced. The PCR reaction system was 5 μL of 2x Hieff® Robust PCR Master Mix (With Dye) (Yixing Biotechnology Co., Ltd.), 0.1 μL of single colony, 0.3 μL of each of UL51-JD-F / R, and 4.3 μL of ddH2O; the PCR program was 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 90 s, for 34 cycles; 72°C for 7 min; 16°C for ∞.

[0040] The results of nucleic acid electrophoresis of the PCR product of the targeted bacteria showed that the theoretical size of the target fragment of the positive targeted bacteria DPV CHv-ΔUL51 was 1759 bp, and PCR identification showed that the fragment size was correct. Combined with the sequencing identification results, it was proved that the UL51 gene of DPV was successfully deleted.

[0041] (3) Second homologous recombination to delete the Kan resistance gene The correct identified clone bacteria were inoculated in 2 mL of LB / Cm and incubated at 30°C and 180 r / min overnight as seed liquid; 10 μL of the seed liquid was added to 1 mL of LB / Cm and incubated at 30°C and 180 r / min for 2 h; 50 μL of 5 mol / L L-arabinose was added and incubated at 30°C and 220 r / min for 1 h; after incubation at 42°C and 180 r / min for 30 min, the bacteria were incubated at 30°C and 180 r / min for 2 h; 1 μL of the bacterial liquid was added to 200 μL of LB and mixed, and then evenly coated on LB / Cm plates and incubated at 30°C for 48 h.

[0042] Several single colonies were picked and correspondingly streaked on LB / Cm+Kan and LB / Cm plates, and incubated at 30°C for 48 h; the colonies that did not grow on the LB / Cm+Kan plate but grew on the LB / Cm plate were identified, and the identification system and procedure were as described above for the identification system and procedure of the duck plague virus UL51 gene deletion colonies, and the nucleic acid sequence was determined.

[0043] The correctly identified colonies were inoculated in 100 mL of LB / Cm and incubated at 30°C and 180 r / min overnight, then centrifuged at 4700 r / min for 10 min, and the supernatant was removed; the bacteria were preserved in 30% glycerol physiological saline and LB containing Cm, and stored in a 80°C refrigerator for standby use.

[0044] The nucleic acid electrophoresis result of the PCR product: the theoretical value of DPV CHv-ΔUL51 removing the Kan resistance gene fragment is 549 bp, the PCR identification result shows that the fragment size is correct, the inserted Kan resistance gene fragment is successfully removed, and the duck plague virus UL51 gene deletion strain infectious clone pDPV CHv-ΔUL51 is obtained.

[0045] Example 2 RFLP identification of UL51 gene deletion The infectious clones of the parent virus, the recovery virus and the deletion virus (denoted as pDPV-CHv50, pDPV CHv-ΔUL51 Rev and pDPV CHv-ΔUL51, respectively) were extracted for plasmid DNA, which was respectively digested with Mlu I and BamH I endonuclease for identification. The RFLP identification system was prepared and incubated at 37°C for 2h, and 1% nucleic acid gel was subjected to constant voltage electrophoresis for 4h.

[0046] The above RFLP identification system is as follows: 1.5μL infectious clone plasmid DNA, 2.5μL 10x Q.CutG. Buffer, 2μL Mlu I or BamH I endonuclease and 19μL ddH2O.

[0047] The RFLP identification electrophoresis result of the infectious clone plasmid after enzyme digestion is shown in Figure 3 It can be seen that BamH there is no difference in the electrophoretic band size after Mlu I enzyme digestion, which is consistent with the theoretical prediction, and the RFLP identification shows that the infectious clone plasmids of DPV-CHv50, DPV CHv-ΔUL51 Rev and DPV CHv-ΔUL51 are correct.

[0048] Example 3 Rescue of UL51 gene deletion strain The bacterial strain of the duck plague virus UL51 gene deletion strain infectious clone which is correct in PCR identification and sequencing was streaked on LB / Cm plate and cultured at 30°C overnight, and a single colony was inoculated into 5mL LB / Cm and cultured at 30°C, 180r / min overnight as seed liquid; the seed liquid was inoculated into 200mL LB / Cm and cultured at 30°C, 180r / min overnight, and the plasmid was extracted and the concentration was determined for standby use.

[0049] DEF cells were prepared with 9-11 day old duck embryos and cultured in 12-well plates at 37℃, 5% CO2 to 80% confluence; the above plasmid was transfected into the DEF cells according to the instructions of the transfection reagent; the cells were cultured at 37℃, 5% CO2, and the presence of fluorescent plaques was observed every day until the cells could not be maintained. The culture wells with fluorescent plaques and cytopathic effects were inoculated into newly prepared DEF cells, and the fluorescence and cytopathic effects were observed every day. When the cells could not be maintained, the samples were collected, 100 μL of DNA was extracted, and PCR and sequencing identification were performed.

[0050] The PCR identification system of the UL51 gene deletion strain of duck plague virus was 5 μL of 2xHieff® Robust PCR MasterMix (With Dye), 0.3 μL of viral DNA, 0.3 μL of UL51-JD-F / R, and 4.1 μL of ddH2O. The identification procedure was the same as that in Example 1 for the duck plague virus UL51 gene deletion colony identification procedure.

[0051] The experimental results are shown in Table 1. Figure 4 As shown in Table 1, fluorescent plaques appeared at 48 h or 72 h after transfection, and the area of the fluorescent plaques increased over time. The corresponding DEF cells showed cytopathic effects, which were the 0th passage (P0). The rescued virus was inoculated into new DEF cells to produce fluorescence and cytopathic effects. The fluorescent area of the virus-infected cells expanded and the cytopathic effects were obvious after 3 passages (P3). After 10 passages (P10) of plaque purification and passage, the cytopathic effects of the virus-infected cells were obvious and the fluorescence disappeared. Therefore, a duck plague virus UL51 gene deletion strain was obtained, which was consistent with the sequencing results, proving that the rescue was successful. The rescue of the duck plague virus UL51 gene recovery virus DPV CHv-ΔUL51 Rev was the same as that of the UL51 gene deletion virus DPV CHv-ΔUL51.

[0052] Example 4: Plaque purification and Western blot identification of the UL51 gene deletion strain The rescued fluorescent DPV CHv-ΔUL51 was inoculated into DEF six-well cell culture plates with 80% confluence and incubated at 37℃, 5% CO2 for 2 h. The culture medium was discarded, and the cells were washed with sterile PBS three times. Then, sterile 2% methylcellulose 1:1 and MEM culture medium were added. The cells were incubated at 37℃, 5% CO2 until cytopathic effects were observed. The sites where cytopathic effects appeared without fluorescence were marked, and several samples from the marked sites were inoculated into DEF cells in 24-well culture plates at 37℃, 5% CO2. The cytopathic effects and fluorescence were observed every day, and the cell wells with cytopathic effects but no fluorescence were selected for sample collection. DNA was extracted, and PCR and sequencing identification were performed. The PCR system and procedure were the same as those in Example 2 for duck plague virus UL51 gene deletion DNA identification.

[0053] After several times of plaque purification, 2~4d obvious cytopathic effect (CPE) of DEF cells inoculated with UL51 gene deletion strain of DPV-CHv-ΔUL51 was observed under white light, but no fluorescence under fluorescence light. The PCR identification results are shown in Fig. 2A, where M is DNA ladder and DEF is DEF cell sample. The PCR and sequencing identification results prove that the UL51 gene deletion strain of DPV-CHv-ΔUL51 completely lacks UL51 gene. Figure 5

[0054] The parental virus DPV-CHv50 rescued from GS1783-pDPV-CHv50, the revert virus DPV CHv-ΔUL51 Rev obtained in the above example and the deletion virus DPV CHv-ΔUL51 were used to infect DEF cells, which were cultured at 37°C, 5% CO2 until CPE was observed, then the cell samples were collected, the supernatant was removed, the samples were lysed and collected using protein lysis solution, the samples were separated by SDS-PAGE, then transferred to PVDF membrane (Millipore, MA, USA), and then the membrane was blocked with 5% skim milk at 37°C for 3h. The membrane was incubated with rabbit anti-UL51 protein (1:500), rabbit anti-ICP8 protein (1:500) and rabbit anti-β-actin polyclonal antibody (1:5000) primary antibody overnight, then washed with PBS containing Tween-20 (PBS-T) three times, then used HRP-labeled goat anti-rabbit IgG (1:5000) as secondary antibody, incubated at room temperature for 1h, and finally used enhanced chemiluminescence kit (ECL) for color development to observe the protein band.

[0055] The WB identification results are shown in Fig. 2B, where UL51 protein expression was detected in DPV-CHv50 and DPV CHv-ΔUL51-Rev infected cells, and no UL51 protein expression was detected in DPV CHv-ΔUL51 infected cells and DEF cells, indicating that the obtained UL51 gene deletion strain of DPV does not express UL51 protein. In this experiment, DPV ICP8 protein was used as the viral internal reference and β-actin was used as the cellular internal reference. Figure 5

[0056] Example 5 Multi-step growth curve of UL51 gene deletion strain ​​The 0.01 MOI of UL51 gene deletion virus DPV CHv-ΔUL51, UL51 gene deletion revertant virus DPV CHv-ΔUL51 Rev and parental virus DPV-CHv50 were inoculated into 12-well plate DEF cells, respectively, and incubated at 37°C, 5% CO2 for 2h, then the supernatant was discarded; washed with sterile PBS for 3 times, added 1 mL of MEM containing 2% NBS to each well, and continued to be cultured at 37°C, 5% CO2. The samples were collected at 24, 48, 60 and 72h, respectively, and the TCID 50 The virus titer was calculated by Reed and Muench method.

[0057] At the same time, the viral DNA of the samples was extracted by DNA genome extraction kit (TIANGEN, Beijing, China), and the DPV genome copy number was determined according to the DPV quantitative PCR method established by the laboratory for DPV UL30 gene (reference Development of TaqMan MGB fluorescent real-time PCR assay for the detection of anatid herpesvirus1 [J]. Virology journal, 2009, 6(1): 71) with UL30-F / R primers and probe UL30-prob in Table 1 and Premix Ex Taq™ premix (Probe qPCR) (Takara, Dalian, China). The qPCR reaction system was 5 μL Premix Ex Taq (2×) (Perfect Real Time), 1 μL DNA, 0.4 μL UL30-probe (SEQ ID NO. 9) probe, 0.4 μL UL30-F (SEQ ID NO. 10), 0.4 μL UL30-R (SEQ ID NO. 11) and 2.8 μL ddH2O; the qPCR program was 95°C, 30s; 95°C, 5s, 60°C, 30s, cycle 40 times.

[0058] The PCR products were quantified by comparison with the standard curve. Statistical analysis was performed using GraphPad Prism version 8 (San Diego, CA, United States), and the data were considered to be significantly different if P value was ≤0.05. * indicates significant difference compared with DPV parental virus (***, P<0.001; **, p<0.01; *, p<0.05).

[0059] The experimental results are shown in Figure 6 TCID 50 The detection showed that Figure 6DPV CHv-ΔUL51, DPV CHv-ΔUL51 Rev and DPV-CHv50 all gradually increased in virus titer over time and reached the peak at 60h, then the virus titer slowly decreased. DPV CHv-ΔUL51 reached the peak at 60h, the average titer was 10 6.16 TCID 50 / 100 μL, which was 0.9 times lower than the average titer of the parent virus 10

[0060] The qPCR detection also showed that the average copy number of DPV CHv-ΔUL51 was 10 Figure 6 / 100 μL, which was 0.9 times lower than the average copy number of the parent virus 10 8.77 / 100 μL. 9.66 / 100 μL.

[0061] The above results showed that the virus titer and copy number of the UL51 gene deletion virus were reduced to different degrees compared with the parent virus, indicating that the UL51 protein would affect the in vitro proliferation of DPV.

[0062] Example 6 Pathogenicity of UL51 gene deletion strain to duck 1. Clinical pathogenicity observation of UL51 gene deletion strain DPV CHv-ΔUL51 to duck 40 14-day-old ducklings were grouped and treated according to Table 2, and the death and mental state of the ducklings were observed and recorded every day until 10d, and the specific experimental process was as shown in Figure 7 A of Table 3.

[0063] Table 2 Grouping and treatment for clinical pathogenicity observation

[0064] The experimental results were as follows: (1) The results of body temperature change were as shown in Figure 7The body temperature of the duck infected with the parent virus DPV CHv50 rose (above 42.5°C) on the first day, peaked (44°C) on the fourth day, then dropped, and gradually returned to normal (40.5-42.5°C) on the sixth day. The body temperature of the duck infected with the revert virus DPV CHv-ΔUL51 Rev rose (above 42.5°C) on the first day, peaked (44°C) on the fourth day, then dropped, and gradually returned to normal (40.5-42.5°C) on the fifth day. The body temperature of the duck infected with the UL51 gene deletion virus DPV CHv-ΔUL51 did not rise obviously, and remained in the normal range (40.5-42.5°C).

[0065] (2) The results of the body weight change are shown in Fig. 2C. Figure 7 As shown in Fig. 2C, the ducks infected with DPV CHv50 and DPV CHv-ΔUL51 Rev showed a trend of weight loss and slow weight gain, with an average weight of about 400 g on the tenth day. The ducks infected with DPV CHv-ΔUL51 showed a similar weight gain trend as the blank control ducks, with an average weight of 700-800 g on the tenth day.

[0066] (3) The results of the mortality rate are shown in Fig. 2D. Figure 8 As shown in Fig. 2D, 6 / 10 of the ducks infected with DPV CHv50 died successively from the second to the sixth day. 8 / 10 of the ducks infected with DPV CHv-ΔUL51 Rev died successively from the second to the seventh day. The ducks infected with DPV CHv-ΔUL51 did not die, like the blank control ducks.

[0067] 2. Organ lesion observation of the duck infected with the UL51 gene deletion strain DPV CHv-ΔUL51 24 14-day-old ducklings were divided into groups and treated according to Table 3. On the fifth and tenth days after infection, 3 ducklings were randomly killed from each group, and the lesions of the organs were observed, photographed, and recorded. Samples were also collected and stored at -80°C for detection of the distribution of the virus in the duck body.

[0068] Table 3. Grouping and treatment for pathological change examination

[0069] The experimental results are as follows: (1) Macroscopic lesions: The parent virus and the revert virus infected ducks had disheveled and dirty feathers, and feces attached around the anus. The UL51 gene deletion virus infected ducks and the MEM group control ducks had no secretions in the mouth, nose, and eyes, and the feathers and anus were clean.

[0070] (2) Necropsy lesions: The results of the fifth day are shown in Fig. 3A. Figure 9As shown in Figure 6, the livers, hearts, glandular stomachs, spleens, intestines, bursa of Fabricius and thymuses of the DPV CHv-ΔUL51 infected ducks and the MEM blank control group ducks had no obvious lesions; the livers, spleens, bursa of Fabricius, duodenum and cecum of the DPV CHv-50 and DPV CHv-ΔUL51Rev infected ducks had severe lesions, and the specific performance was that the livers and spleens of the ducks were dark, the bursa of Fabricius, duodenum and cecum wall were thin, and there was severe hemorrhage, and the thymus was hemorrhagic and atrophic.

[0071] 3. Distribution of UL51 gene deletion strain DPV CHv-ΔUL51 in duck body The tissue samples of the above experimental group 5d were extracted to obtain DNA, and the qPCR method in Example 5 was used to detect the DPV genome copy number of different organs at different infection times of different groups.

[0072] The experimental results are shown in Figure 7. Figure 10 As shown in Figure 7, the average copy number of DPV CHv-ΔUL51 in 100 mg of tissue at 5d was: liver 10 3.57 , heart 10 3.94 , glandular stomach 10 4.05 , spleen 10 4.1 , bursa of Fabricius 10 4.72 , duodenum 10 3.82 , cecum 10 3.98 , thymus 10 4.2 ; the average copy number of DPV CHv-CHv50 and DPV CHv-ΔUL51Rev in 100 mg of tissue at 5d was: liver 10 6.25 / 6.24 , heart 10 6.97 / 8.11 , glandular stomach 10 7.71 / 8.16 , spleen 10 8.86 / 9.04 , bursa of Fabricius 10 8.68 / 9.46 , duodenum 10 7.39 / 8.27 , cecum 10 7.09 / 8.95 , thymus 10 9.93 / 10.08 .

[0073] The DPV genome load of the UL51 gene deletion strain infected duck at 5d in each tissue organ was significantly lower than that of the parent virus and the reverse virus, indicating that the deletion of the UL51 gene significantly reduced the proliferation of DPV in the duck body.

[0074] 4. UL51 gene deletion strain DPV CHv-ΔUL51 immune challenge protection experiment Sixty-four 14-day-old ducklings were grouped and immunized according to Table 4, and 14 days after immunization, 100 LD 50The ducks were challenged with the virulent DPV CHv virus. In Group I of each group, the rectal temperature and weight of the ducks were measured and recorded daily, and the mortality and mental state of the ducks were observed and recorded for 10 days. In Group II of each group, 3 ducks were randomly euthanized on days 5 and 10 after challenge with the virus, and the lesions of each tissue and organ were observed and photographed.

[0075] Table 4. Experimental Groups for Immunization / Challenge Protection and Immunization / Challenge

[0076] The experimental results are as follows: (1) Changes in body temperature: Results are shown in Figure 10 As shown in Figure A, the body temperature of the MEM blank control ducks began to rise on day 2 after being challenged with the virulent DPV virus, with some ducks reaching a temperature of 42.5℃. On day 4, all ducks maintained a high fever until death. 5 TCID 50 and 10 4 TCID 50 Like ducks immunized with the duck plague attenuated live vaccine, ducks immunized with the UL51 gene deletion strain had their body temperature fluctuate within the normal range (41.0–42.5℃) throughout the experiment.

[0077] (2) Weight changes: Results are shown in Figure 10 As shown in B, the weight gain of the MEM control ducks after being challenged with the virulent DPV virus showed a slowing trend, which continued until all the ducks died; 10 5 TCID 50 and 10 4 TCID 50 Like ducks immunized with the duck plague attenuated live vaccine, ducks immunized with the UL51 gene deletion strain maintained a consistent weight gain throughout the experiment and exhibited a similar growth trend.

[0078] The above results indicate that the UL51 gene-deleted strain DPV CHv-ΔUL51 can protect ducks from fever and maintain stable body temperature under lethal challenge with virulent DPV CHv. Furthermore, 10 4 TCID 50 The DPV CHv ΔUL51 immunization of ducks against virulent DPV attack achieves the same protective effect as a duck plague attenuated live vaccine, protecting ducks at 100 LD. 50 Under DPV (Disease-Produced Virus) CHv attack, the individual does not die, develop fever, or lose weight.

[0079] (3) Mortality rate: Results are shown in Figure 11 As shown in C, 10 5 TCID 50 and 10 4 TCID 50Ducks immunized with the UL51 gene-deleted strain, like those immunized with the duck plague attenuated live vaccine, showed no mortality, with a survival rate of 10 / 10, indicating a 100% protection rate. In contrast, all ducks in the MEM control group died after challenge with the virulent DPV virus. This demonstrates that immunization with the UL51 gene-deleted strain resulted in 10 / 10 survival rates. 4 TCID 50 The UL51 gene deletion strain has the same effect as immunizing one dose of commercially available vaccine, providing 100% protection against lethal DPV attacks in ducks.

[0080] (4) Autopsy findings: Autopsy results after 5 days are as follows ​ As shown: 10 5 TCID 50 No obvious lesions were observed in any organs of ducks immunized with DPV CHv-ΔUL51 and duck plague attenuated live vaccine after challenge; 10 4 TCID 50 Ducks immunized with DPV CHv-ΔUL51 showed no obvious organ lesions after challenge, although some ducks exhibited thymus atrophy and hemorrhage.

[0081] Five days after being challenged with a strong MEM virus, the control ducks exhibited severe hemorrhage in the liver, sloughing of the proventricular mucosa with a clear hemorrhagic ring at the junction with the esophagus, hemorrhage in the bursa of Fabricius, hemorrhage in the duodenum and cecum, sloughing of the intestinal mucosa, thinning of the intestinal wall, hemorrhage in the spleen with pinpoint necrotic foci, and necrotic foci in the thymus.

[0082] It is evident that the UL51 gene-deleted strain DPV CHv-ΔUL51, similar to ducks immunized with the duck plague attenuated live vaccine, inhibited the proliferation of the virulent DPVCHv virus in ducks after challenge, with no obvious lesions in any tissues or organs. In contrast, when unimmunized ducks were challenged with the virulent DPVCHv virus, the virus rapidly multiplied in their bodies, causing severe lesions in various tissues and organs, ultimately leading to the death of the ducks. The prepared UL51 gene-deleted strain DPV CHv-ΔUL51 has the potential to serve as an attenuated live vaccine for the prevention of duck plague, and can be used in duck farming and disease control.

[0083] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A duck plague virus UL51 gene deletion strain DPV CHv-ΔUL51 (DPV CHv-ΔUL51) Mardivirus ), characterized in that, The gene deletion strain has been deposited in the China Center for Type Culture Collection of Wuhan University on April 2, 2025, and the preservation number is CCTCC NO: V202523.

2. The application of the gene deletion strain of claim 1 in the research of the pathogenicity mechanism of duck plague virus affected by the UL51 gene and the protein coded by the UL51 gene.

3. The application of the gene deletion strain of claim 1 in the preparation of attenuated vaccine of duck plague virus.

4. The application of the gene deletion strain of claim 1 in the field of duck breeding, live vector vaccine and multiple vaccine.

5. Use according to claim 4, characterized in that, The attenuated vaccine prepared by the UL51 gene deletion strain can be used for the prevention of duck plague.

6. The method for constructing a duck plague virus UL51 gene deletion strain according to claim 1, wherein, Comprising the following steps: (1) Obtaining a targeting fragment PCR amplification is performed on the pEP-Kan-S plasmid as a template and the primers with nucleotide sequences as shown in SEQ ID NO. 1~2 to obtain a UL51 gene deletion targeting fragment; (2) First homologous recombination to delete the target gene The obtained UL51 gene deletion targeting fragment is added to the competent bacteria, mixed, and then subjected to electroporation and plating. Single colonies are picked as templates, and single colony PCR identification is performed using primers with nucleotide sequences as shown in SEQ ID NO. 7~8 to screen positive clones in which the Kan resistance gene replaces the UL51 gene; (3) Second homologous recombination to delete the Kan resistance gene The positive clone bacteria obtained in step (2) are inoculated into LB / Cm, cultured at 30℃, 180r / min overnight as seed liquid; the seed liquid is added to LB / Cm, cultured at 30℃, 180r / min; L-arabinose is added, cultured at 30℃, 220r / min; cultured at 42℃, 180r / min, and then cultured at 30℃, 180r / min; the bacterial liquid is mixed with LB and then plated on LB / Cm plates and cultured at 30℃; several single colonies are picked and plated on LB / Cm+Kan and LB / Cm plates respectively and cultured at 30℃; colonies that do not grow on the LB / Cm+Kan plate but grow on the LB / Cm plate are selected for PCR identification using primers with nucleotide sequences as shown in SEQ ID NO. 7~8. The correct bacteria are the clone bacteria with the duck plague virus UL51 gene deletion strain infectious clone pDPV CHv-ΔUL51; (4) Virus rescue The pDPV CHv-ΔUL51 plasmid DNA is extracted and transfected into DEF cells. When cytopathic effect occurs, the culture sample is collected and inoculated into fresh DEF cells to obtain the UL51 gene deletion virus DPV CHv-ΔUL51.

7. The construction method of claim 6, wherein, In step (1): The PCR amplification system is: PrimeSTAR Max DNA Polymerase, primers with nucleotide sequences as shown in SEQ ID NO. 1~2, pEP-Kan-S plasmid, and ddH2O; The PCR amplification program is: 98℃, 2min; 98℃, 10s, 55℃, 15s, 72℃, 5s, a total of 34 cycles; 72℃, 10min; 16℃, ∞.

8. The construction method of claim 6, wherein, In step (2), the competent bacteria are GS1783-pDPV-CHv50; the preparation method is as follows: The GS1783-pDPV-CHv50 strain is streaked on an LB / Cm plate and cultured at 30 DEG C overnight; a single colony is inoculated into LB / Cm and cultured at 30 DEG C and 180 r / min overnight as a seed liquid; The seed liquid is inoculated into LB / Cm and cultured at 30 DEG C and 220 r / min until the OD is 0.5, then transferred to 42 DEG C and 220 r / min and immediately placed in an ice bath for standing; centrifuged at 4 DEG C to remove the supernatant, the bacterial body is blown apart with 4 DEG C pre-cooled sterilized ddH2O, centrifuged at 4 DEG C to remove the supernatant, and ddH2O is added to obtain the competent bacteria.

9. The construction method of claim 6, wherein, In step (2), The single colony PCR identification system is as follows: 2xHieff Robust PCR Master Mix, single colony, and primers UL51-JD-F / R with the nucleotide sequence shown in SEQ ID NO. 7-8, and ddH2O; The PCR identification procedure is as follows: 94 DEG C, 5 min; 94 DEG C, 30 s, 55 DEG C, 30 s, 72 DEG C, 90 s, cycle 34 times; 72 DEG C, 7 min; 16 DEG C, ∞.

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