Recombinant porcine epidemic diarrhea virus with enhanced replication capability

Abstract

The invention relates to animal virology and genetic engineering technologies, and aims to provide a recombinant porcine epidemic diarrhea virus with enhanced replication capability. The virus is obtained by modifying a porcine epidemic diarrhea virus PEDV, and the amino acid sequence of nucleocapsid protein (N protein) of the virus is shown as SEQ ID NO: 2. The strain of the virus is preserved in the China Center for Type Culture Collection, the preservation name is rcPEDV P120-NT139A, and the preservation number is CCTCC NO: V202562. According to the invention, threonine at the 139th site of nucleocapsid protein of PEDV is a key modification site influencing PEDV infection, and the new strain can be successfully infected in PEDV infection model verification experiments of IPEC-J2 and Vero cells; the strain shows stronger replication ability in a PEDV infection model verification experiment of IPEC-J2 cells, the production difficulty is reduced, and the strain can be further used for preparing a porcine epidemic diarrhea virus inactivated vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal virology and genetic engineering, and particularly relates to a novel recombinant porcine epidemic diarrhea virus with enhanced replication capacity. BACKGROUND

[0002] Porcine epidemic diarrhea virus (PEDV) causes porcine epidemic diarrhea (PED), which has recently broken out and spread widely in the world. PEDV mainly infects pig enterocytes, colonizes in the intestinal tract, causes intestinal villi atrophy, necrosis and shedding, thereby affecting the absorption of nutrients, and leading to vomiting, diarrhea, weight loss, anorexia and depression. PEDV has caused great public health threat and economic burden to the pig industry, and has become one of the most important pathogens threatening the pig industry. Therefore, it is of great significance to clarify the infection regulation, immune escape and pathogenesis of the virus for the prevention and control of the disease.

[0003] PEDV belongs to the coronavirus family of the alpha coronavirus genus, is a single-stranded positive-sense RNA virus wrapped by a capsid, and has a total length of about 28 kb, encoding four structural proteins including spike protein (S, 180-220 kDa), membrane protein (M, 27-32 kDa), envelope protein (E, 7 kDa) and nucleocapsid protein (N, 55-58 kDa), and 17 non-structural proteins (nsp1-nsp16 and ORF3), in the order of 5'-ORF1a / 1b-S-ORF3-E-M-N-3'. Various protein structures of PEDV play different important functions, and post-translational modification (PTM) of proteins has important influence on the structure, activity and function of proteins. PTM includes acetylation, phosphorylation, ubiquitination, etc., and plays an important role in immune protection, virus replication, inflammation, etc. It has been reported that proteins such as BST2, PABPC4 and TRIM21 of host cells can regulate ubiquitination modification of PEDV N protein, thereby causing degradation of N protein and inhibiting replication of PEDV. Therefore, design and use of recombinant viruses for key post-translational modification sites of viral proteins are also an important research idea and means for prevention and control of PEDV epidemic and spread.

[0004] At present, vaccines for preventing and controlling PEDV mainly include inactivated vaccines, subunit vaccines, attenuated live vaccines and nucleic acid vaccines, but there are still deficiencies such as insufficient cross-protection of multiple genotypes, insufficient mucosal immune induction and interference of maternal antibodies, resulting in inability to produce effective protection. Therefore, it is necessary to adjust the new idea of inhibiting PEDV epidemic and propose new solutions. SUMMARY

[0005] The technical problem solved by the present application is to overcome the deficiencies in the prior art and provide a recombinant porcine epidemic diarrhea virus with enhanced replication capacity.

[0006] To solve the technical problem, the solution of the present application is:

[0007] The present application provides a recombinant porcine epidemic diarrhea virus with enhanced replication capacity, and the virus strain is deposited in the China Center for Type Culture Collection, with the deposit name rcPEDV P120-N T139A , and the deposit number is CCTCC NO: V202562.

[0008] As a preferred solution of the present application, the virus is obtained by modification of porcine epidemic diarrhea virus PEDV, and the amino acid sequence of the nucleocapsid protein (N protein) is shown in SEQ ID NO: 2.

[0009] The present application further provides a preparation method of the aforementioned recombinant porcine epidemic diarrhea virus, comprising: taking the threonine at position 139 in the nucleocapsid protein sequence of porcine epidemic diarrhea virus PEDV as a target, and mutating the target to alanine.

[0010] As a preferred solution of the present application, the preparation method specifically comprises:

[0011] (1) constructing a recombinant plasmid pCMV-PEDV N-HA based on the PEDV N protein sequence, and mutating the threonine at position 139 (N-T139) in the N protein of PEDV to alanine;

[0012] (2) based on the recombinant plasmid pCMV-PEDV N-HA, rescuing the recombinant virus and expanding it to obtain a virus strain capable of stable proliferation on passage cells.

[0013] The present application also provides a biological product containing the aforementioned recombinant porcine epidemic diarrhea virus, and the biological product is used for preventing and / or treating porcine epidemic diarrhea virus disease.

[0014] As a preferred solution of the present application, the biological product is a live vaccine or an inactivated vaccine, and further comprises a pharmaceutically acceptable adjuvant.

[0015] As a preferred solution of the present application, the biological product is a medicine, and further comprises a pharmaceutically acceptable adjuvant.

[0016] Invention principle description:

[0017] In view of the actual application effect of the existing prevention PEDV research results in the industry is not ideal, the present application proposes to design a new type of recombinant virus for enhancing the replication of porcine epidemic diarrhea virus, and provides an alternative new vaccine strain.

[0018] Applicant noted in the research process that the nucleocapsid (N) protein in the structural protein of PEDV is highly conserved, is the protein with the highest expression abundance, and forms a complex with genomic RNA, thereby affecting the synthesis, packaging of viral RNA and assembly of virus particles. The prediction of PEDV N protein also shows that there are multiple potential acetylation and phosphorylation modification residues, and the influence on PEDV replication is explored by mutating these residues, thereby providing a new strain and target for the development of a new type of PEDV vaccine and antiviral drugs, and there is no public report on the inactivated vaccine obtained by using a reverse genetics system based on the phosphorylation modification site of the N protein in the present stage of public literature.

[0019] Based on the research results of the performance of the N protein, the research team of the applicant clarified the infection regulation mechanism of PEDV, and innovatively proposed to take the acetylation / phosphorylation modification residues of the specific target site in the N protein of PEDV as the modification object, thereby providing a new idea for vaccine development. In the existing research results, there is no explicit record about using the nucleocapsid (N) protein and the acetylation / phosphorylation modification residues of the specific target site to construct a recombinant virus.

[0020] The applicant has conducted in-depth research and complex identification on the PEDV N protein, and finally confirmed that the threonine at the 139th position of the nucleocapsid protein (N-T139) regulates the replication ability of PEDV, and on this basis, further proposed the following recombinant virus construction design scheme:

[0021] (1) Based on the sequence characteristics of the N protein of PEDV, it is found through immunoprecipitation (Co-IP) and mass spectrometry identification results that the threonine at the 139th position can be phosphorylated, and therefore the threonine at the 139th position is designed to be mutated.

[0022] (2) After determining the modification site, based on the characteristics that the side chain structure of alanine is simple and can make the function of the site fail, the threonine at the 139th position is mutated to alanine (T139A).

[0023] Through verification in a PEDV natural infection model, the results prove that the rcPEDV P120-N T139A The replication ability of the recombinant virus is significantly enhanced.

[0024] Compared with the prior art, the beneficial effects of the present application are:

[0025] 1. The present application finds the amino acid site subjected to post-translational modification in the PEDV N protein through in-depth research, and determines that it is a key site affecting virus replication, and uses the characteristics that the site is highly conserved in different subtypes of porcine epidemic diarrhea virus, and proposes to take it as a new target for broad-spectrum anti-porcine epidemic diarrhea virus drugs and attenuated vaccines for porcine epidemic diarrhea virus.

[0026] 2, The present application further provides a new recombinant PEDV strain based on a new target, which can successfully infect in PEDV infection model verification experiments of IPEC-J2 and Vero cells, and the strain shows stronger replication capacity in the PEDV infection model verification experiment of IPEC-J2 cells, reduces the production difficulty, and can be further used for preparing porcine epidemic diarrhea virus inactivated vaccine.

[0027] 3, The present application determines that the threonine at position 139 of the nucleocapsid protein of PEDV is a key modification site affecting PEDV infection, and on this basis, a recombinant virus targeting this site can improve the replication level of the virus. The present application further enriches the research strategy for PEDV infection regulation and pathogenesis mechanism, and provides a new idea for developing new prevention and control methods.

[0028] 4, The present application provides a new research and development idea and corresponding technical inspiration for scientific researchers to further understand the virus infection regulation rule and find new clues for prevention and control in the research work of other similar coronaviruses. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is the mass spectrum identification result for PEDV N protein.

[0030] Figure 2 is the WB identification result of immunoprecipitation for PEDV N protein phosphorylation modification.

[0031] Figure 3 is the N protein structure description in the present application. T139A

[0032] Figure 4 is the rcPEDV P120-N recombinant virus identification result in the present application. T139A

[0033] Figure 5 is the rcPEDV P120-N recombinant virus infection IPEC-J2, Vero cell result. T139A

[0034] Figure 6 is the Western Blot and qPCR result of rcPEDV P120-N recombinant virus replication capacity. T139A

[0035] Figure 7 is the IFA result of rcPEDV P120-N recombinant virus replication capacity. T139A

[0036] Figure 8 is the rcPEDV P120-N​​​​​T139A Growth curve of recombinant virus. DETAILED DESCRIPTION

[0037] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application. The present application is not limited to the following examples, and the test methods not described in detail in the examples are conventional methods in the art.

[0038] Example 1 Screening of threonine at position 139 of PEDV N protein as a modification target

[0039] PEDV N protein sequences (GenBank: MK409659.1) were downloaded from the NCBI database to construct recombinant plasmid pCMV-PEDV N-HA. Samples were collected through in vitro overexpression experiments and animal experiments, and mass spectrometry was performed through Co-IP experiments to confirm that the N protein can be phosphorylated and modified, and the modification site is located at threonine at position 139. Figure 1 The mass spectrometry results confirmed that the threonine at position 139 of the PEDV N protein selected by the present application can be used as a modification target. Figure 2 The identification results of the phosphorylation antibody immunoprecipitation are shown. As can be seen from the figure, the PEDV N protein can be recognized and combined by the phosphorylation antibody after overexpression, and the black arrow in the figure points to the protein band at the position of 55 kDa (the size position of PEDV N protein), which indicates that the PEDV N protein is phosphorylated and modified.

[0040] Example 2 rcPEDV P120-N T139A Recombinant virus infection model

[0041] (1) Construction of a novel recombinant virus targeting PEDV N protein T139A

[0042] According to the results in Example 1, it is determined that threonine at position 139 of the PEDV N protein (N-T139) can be phosphorylated and modified. According to the description in the principle part, the threonine is designed, and then the threonine at this site is mutated to alanine (A). Figure 3

[0043] The specific sequence of the PEDV N protein is shown below. The underlined abbreviations T and A in the sequence are threonine and alanine, respectively, which is mutated from threonine.

[0044] SEQ ID NO: 1 (virus name rcPEDV P120), N protein amino acid sequence: ​MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNLGVRKASEKPIIPNFSQQLPSVVEIVEPN T PPTSRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSRNRGNNQGRGASQNRGGNNNNNNKSRNQSKNRNQSNDRGGVTSRDDLVAAVKDALKSLGIGENPDKLKQQQKPKQERSDSSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKPQRKKEKKNKRETTQQLNEDAIYDDVGLPSDSTHANLEWDTAVDGGDTAVEIINEIFDTGN; SEQ ID NO: 2 (virus name rcPEDV P120-N T139A ), N protein amino acid sequence: MASVSFQDRGRKRVPLSLYAPLRVTNDKPLSKVLANNAVPTNKGNKDQQIGYWNEQIRWRMRRGERIEQPSNWHFYYLGTGPHADLRYRTRTEGVFWVAKEGAKTEPTNLGVRKASEKPIIPNFSQQLPSVVEIVEPN A PPTSRANSRSRSRGNGNNRSRSPSNNRGNNQSRGNSRNRGNNQGRGASQNRGGNNNNNNKSRNQSKNRNQSNDRGGVTSRDDLVAAVKDALKSLGIGENPDKLKQQQKPKQERSDSSGKNTPKKNKSRATSKERDLKDIPEWRRIPKGENSVAACFGPRGGFKNFGDAEFVEKGVDASGYAQIASLAPNVAALLFGGNVAVRELADSYEITYNYKMTVPKSDPNVELLVSQVDAFKTGNAKPQRKKEKKNKRETTQQLNEDAIYDDVGLPSDSTHANLEWDTAVDGGDTAVEIINEIFDTGN; In view of the stronger virus transmission and replication ability of PEDV P120 and the ease of spreading between cells, the venom of PEDV P120 is selected when constructing a recombinant virus.

[0045] As an example, the present application uses a method for constructing a porcine epidemic diarrhea virus infectious cDNA plasmid in one step to rescue the virus. The specific operation of the method can refer to the literature recorded by Jang, Guehwan et al. (doi:10.3390 / v14112319), and is briefly described as follows:

[0046] In this experiment, the RNA of PEDV P120 strain was first obtained and reversely converted into total cDNA as a template, then subfragment amplification and purification, molecular marker site modification were carried out, and then pBeloBAC11 system was used for construction of infectious clone plasmid. After sequencing the positive clones, the clones with correct sequencing were preserved and the plasmid was extracted and stored at-20℃. Finally, the rescued recombinant virus was rescued and propagated to the required passage.

[0047] Further, the present application uses full-length nucleotide sequencing to verify the sequence of the rescued recombinant virus, and the results are shown in Figure 4 . As can be seen from the figure, the 566th adenine deoxyribonucleotide (A) is successfully mutated into guanine deoxyribonucleotide (G), indicating that the strain carries the correct molecular marker, and the virus rescued in this experiment is indeed the recombinant virus rcPEDV P120-N T139A .

[0048] The applicant preserves the strain of the recombinant porcine epidemic diarrhea virus, and the preservation information is as follows: the preservation name is rcPEDV P120-N T139A , the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation address is Wuhan, Wuhan University, China, the preservation number is CCTCC NO: V202562, and the preservation time is August 28, 2025.

[0049] (2) rcPEDV P120-N T139A Recombinant virus infects IPEC-J2 and Vero cells

[0050] The rescued recombinant virus is propagated to the 4th passage (P4), and the titer (TCID 50 ) is determined. IPEC-J2 cells and Vero cells are infected at MOI=1, and after 4 h of infection adsorption, the medium is replaced with a medium containing 4 μg / mL trypsin and maintained for 20 h. The results show that the rcPEDV P120-N T139A recombinant virus can infect IPEC-J2 and Vero cells Figure 5). It can be seen from the figure that the group added with recombinant virus showed obvious cytopathic effect (CPE), indicating that both IPEC-J2 cells and Vero cells could be infected by rcPEDV P120-N T139A .

[0051] Example 3 rcPEDV P120-N T139A Exploration of replication ability of recombinant virus

[0052] (1) Verification of rcPEDV P120-N T139A recombinant virus replication ability

[0053] According to the operation in Example 2, IPEC-J2 cells and Vero cells were infected at MOI = 1, and blank (Mock) wells, wild (rcPEDV P120) wells and mutant (rcPEDV P120-N T139A ) wells were set. The cells were lysed to extract total cell protein and total cell RNA at 24 h after infection, and the expression of virus genes and proteins in the cells was detected by qPCR and Western-Blot methods. The results showed that the replication ability of rcPEDV P120-N T139A was better than that of wild-type rcPEDV P120 recombinant virus Figure 6 . It can be seen from the figure that the PEDV N and S1 protein expression of rcPEDV P120-N T139A recombinant virus group was higher than that of rcPEDV P120 group, and the transcription level of PEDV N and ORF1 gene was also higher.

[0054] Consistent results were also shown in IFA test Figure 7 . It can be seen from the figure that the fluorescence intensity of PEDV N protein of rcPEDV P120-N T139A recombinant virus group was stronger than that of rcPEDV P120 group.

[0055] (2) Growth curve of rcPEDV P120-N T139A recombinant virus

[0056] Vero E6 cells were plated in 7 twenty-four-well plates about 12 h in advance, at a density of about 1.5*10 5 / mL, 250 μL / well, and 2 wells were plated for each plate. The cells were infected when they grew to about 80%. The amount of virus was calculated at MOI = 0.05, and the rcPEDV-P120, rcPEDV P120-N T139ADilution was performed, and each was configured with 4 mL of virus liquid. Vero E6 cells were washed twice with D-Hanks, the waste liquid was discarded, and the prepared virus liquid was added. Starting from the time when the virus liquid was added, a twenty-four-well plate was collected at 8, 16, 24, 32, 40, and 48 h, respectively, and was directly frozen at-80℃. After thawing at room temperature, the liquid in the well was transferred to a 1.5 mL EP tube, vortexed thoroughly, centrifuged at 12,000 rpm for 3 min, and placed on ice. The virus titers at each time point were determined, and the growth curve was drawn. The results show that the rcPEDV P120-N T139A The replication speed of the recombinant virus is better than that of the wild-type rcPEDV P120 recombinant virus Figure 8 It can be seen from the figure that the titers of the rcPEDV P120-N T139A The titers of the recombinant virus are higher than those of the rcPEDV-P120 group.

[0057] Analysis and conclusion:

[0058] Based on the results of Examples 1 and 2, it can be seen that the rcPEDV P120-N T139A The replication ability of the recombinant virus is better than that of the wild-type rcPEDV P120 recombinant virus; the recombinant virus can infect susceptible cells such as IPEC-J2 and Vero, and virus particles can be detected at 12 h after infection. Therefore, the recombinant virus can be further used for animal experiments to explore its prospects as an inactivated vaccine, and to achieve the inhibition of PEDV virus prevalence.

[0059] A specific application method is as follows: the recombinant porcine epidemic diarrhea virus is used to prepare a biological product for preventing and / or treating porcine epidemic diarrhea virus disease. The biological product can be a live vaccine or an inactivated vaccine, and also includes a pharmaceutically acceptable adjuvant. Alternatively, the biological product is a drug, and also includes a pharmaceutically acceptable adjuvant.

[0060] The preparation process and use method of the live vaccine, inactivated vaccine, or drug all refer to the existing technology for preparing a live vaccine, inactivated vaccine, or drug using a porcine epidemic diarrhea virus biological product.

[0061] The scope of protection of the application is not limited to the specific embodiments described herein, but also encompasses any alternatives, modifications, equivalents and / or improvements thereto within the spirit and principle of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. It will be apparent to those skilled in the art that various modifications and variations can be made to the specific embodiments without departing from the spirit or scope of the application. There is no intention to limit the application other than as by the appended claims. Any and all modifications, variations or equivalent arrangements that fall within the scope of the application along with alternatives, modifications, equivalents and / or improvements thereto are intended to be included within the scope of the application.

Claims

1. A recombinant porcine epidemic diarrhea virus with enhanced replication capacity, characterized in that, The strain of the virus is preserved in China Center for Type Culture Collection, and the preservation name is porcine epidemic diarrhea virus rcPEDV P120-N T139A , and the preservation number is CCTCC NO: V202562.

2. The recombinant porcine epidemic diarrhea virus according to claim 1, characterized in that, The virus is obtained by modification of porcine epidemic diarrhea virus PEDV, and the amino acid sequence of the nucleocapsid protein is shown as SEQ ID NO:

2.

3. The method of producing a recombinant porcine epidemic diarrhea virus according to claim 1, characterized in that, The application relates to a biological product for preventing and / or treating porcine epidemic diarrhea virus disease. The threonine at the 139th position in the nucleocapsid protein sequence of the porcine epidemic diarrhea virus PEDV is taken as a target, and the target is mutated into alanine.

4. The method of claim 3, wherein, Specifically, the application comprises the following steps: (1) constructing a recombinant plasmid pCMV-PEDV N-HA based on the PEDV N protein sequence, and mutating the threonine at the 139th position in the N protein of the PEDV into alanine; (2) rescuing a recombinant virus based on the recombinant plasmid pCMV-PEDV N-HA, and expanding and propagating the virus to obtain a virus strain capable of stably proliferating on a passaged cell.

5. A biological product comprising the recombinant porcine epidemic diarrhea virus of claim 1, characterized in that, The biological product is used for preventing and / or treating porcine epidemic diarrhea virus disease.

6. The biological according to claim 5, characterized in that, The biological product is a live vaccine or an inactivated vaccine, and further comprises a pharmaceutically acceptable adjuvant.

7. The biological product of claim 5, wherein, The biological product is a medicine, and further comprises a pharmaceutically acceptable adjuvant.

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