IPEC-J2 cell strain capable of stably expressing PED virus nucleocapsid protein truncated fragment and application of IPEC-J2 cell strain
By designing IPEC-J2 cell lines that stably express the truncated fragment of PED viral nucleocapsid protein, the problem of poor proliferation of PEDV in IPEC-J2 cells was solved, and the viral replication ability was significantly enhanced, and it was used for the study of virus isolation and infection mechanisms.
Patent Information
- Application Number
- CN202510227522.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-06-06
AI Technical Summary
In the prior art, the proliferation effect of swine epidemic diarrhea virus (PEDV) in IPEC-J2 cells is poor, limiting the progress of virus isolation, passage and infection mechanism research.
By designing an IPEC-J2 cell line that stably expresses the nucleocapsid protein truncated fragment of PED viral, the cell line was able to grow normally in a culture environment of 10 μg/mL puromycin, and cell lines carrying the truncated fragment of PEDV N protein were obtained through lentiviral packaging and puromycin screening.
This cell line significantly enhances the replication ability of PEDV, can be used for virus isolation, passage and study PEDV infection regulation mechanisms, providing clues to new prevention and control methods.
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Figure CN120098932A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bioengineering, and specifically relates to an IPEC-J2 cell strain capable of stably expressing a truncated fragment of a nucleocapsid protein of porcine epidemic diarrhea virus and an application thereof. Background Art
[0002] Porcine epidemic diarrhea (PED) has been common in recent years and is the main intestinal disease that causes fatal watery diarrhea in newborn piglets. Porcine epidemic diarrhea virus (PEDV) is the pathogen of the disease, which mainly infects the villi and crypt epithelial cells and goblet cells in the small intestine, causing villous atrophy, malabsorption, diarrhea, vomiting, and anorexia. PEDV is a member of the genus Alphacoronavirus. Its genome is about 28kb long and consists of a 5' untranslated region (UTR), 7 open reading frames (ORF) and a 3'UTR. It encodes 4 structural proteins, namely spike (S) protein, envelope (E) protein, membrane (M) protein, nucleocapsid (N) protein, 16 non-structural proteins (Nsp 1-16) and 1 auxiliary protein ORF3. Among them, the nucleocapsid protein is a highly conserved protein that wraps the viral RNA genome, affects viral replication and transcription, accounts for the largest proportion among structural proteins, and is expressed in large quantities in infected cells.
[0003] IPEC-J2 cells are a cell line isolated from the jejunum of unsuckling newborn piglets. After infection with PEDV, the expression profile of its cytokines is similar to the response of target cells in vivo, which can simulate the natural infection of PEDV in vivo and is an optimal cell model for PEDV-related research. In addition, the isolation of certain clinical strains may rely on host cells or specific conditions to adapt and replicate effectively. Strains isolated and propagated in host cells are more conducive to maintaining their original characteristics. In recent years, scientists have usually used Vero cells for the isolation and propagation of PEDV, mainly based on the advantages of the cell's good susceptibility to PEDV, stability, good safety, and the ability to produce high-titer viruses. However, the currently isolated IPEC-J2 cells can only be used to study the PEDV infection regulation mechanism due to their poor infection effect, and cannot be used for virus isolation, propagation, etc.
[0004] Given that in actual applications, the poor proliferation effect of PEDV in IPEC-J2 cells has limited the above practices or research results, it is urgent to establish an IPEC-J2 cell line with good adaptability to PEDV for virus isolation and propagation, to reveal the PEDV infection mechanism and host response, and thus provide clues for finding new prevention and control measures. Summary of the invention
[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide an IPEC-J2 cell line stably expressing a truncated fragment of the PED virus nucleocapsid protein and its application; the cell line can be used to enhance the replication of porcine epidemic diarrhea virus.
[0006] To solve the technical problem, the solution of the present invention is:
[0007] Provided is an IPEC-J2 cell line that stably expresses a truncated fragment of a PED virus nucleocapsid protein. The cell line is deposited in the China Center for Type Culture Collection, with the deposited name being porcine small intestinal epithelial cell line R58-WT-IPEC, the Latin name being Susscrofa, the deposited number being CCTCC NO: C202425, and the deposited date being October 24, 2024.
[0008] As a preferred embodiment of the present invention, the cell line is a stably transfected cell line targeting the replication stage of PEDV, and can stably express the target fragment sequence shown in SEQ ID NO: 1 in the PEDV nucleocapsid protein.
[0009] As a preferred embodiment of the present invention, the cell line has both a fluorescent tag protein (CopGFP) and a puromycin resistance gene (Puro), the sequence of the former is shown in SEQ ID NO:2, and the sequence of the latter is shown in SEQ ID NO:3.
[0010] The present invention further provides a method for constructing the aforementioned IPEC-J2 cell line, comprising the following steps:
[0011] (1) Vector construction: The target truncated fragment of PEDV N protein was inserted into the CD513B-1 vector by homologous recombination; after transformation and sequencing, a lentiviral recombinant plasmid was obtained;
[0012] (2) Lentiviral packaging: The lentiviral recombinant plasmid and packaging plasmids pMD2.G, pMDlgpRRE, and pRSV-Rev were co-transfected into 293T cells to obtain viral fluid;
[0013] (3) Infecting IPEC-J2 cells with lentivirus: Increasing the amount of lentivirus infection in IPEC-J2 cells by repeated infection;
[0014] (4) Screening of stable cell lines: Positive cell lines were screened by adding puromycin and gradually increasing the concentration to obtain the IPEC-J2 cell line carrying the truncated fragment of the PEDV N protein.
[0015] As a preferred embodiment of the present invention, the sequence of the CD513B-1 vector is shown in SEQ ID NO:4.
[0016] As a preferred embodiment of the present invention, the IPEC-J2 cell line carrying the truncated fragment of the PEDV N protein can grow normally in a culture environment of 10 μg / mL puromycin.
[0017] The present invention provides the use of the aforementioned IPEC-J2 cell strain in promoting PEDV replication.
[0018] The present invention also provides the use of the aforementioned IPEC-J2 cells in the preparation of porcine epidemic diarrhea virus vaccines and medicines.
[0019] Description of the invention principle:
[0020] In view of the fact that the existing work results on the isolation, propagation and virus infection mechanism research of PEDV virus in the industry are not ideal, the present invention proposes a new solution, which proposes to enhance the replication of the virus on IPEC-J2 cells by designing a cell line for enhancing the replication ability of PEDV.
[0021] During the research process, the applicant noticed that the PEDV nucleocapsid protein forms a complex with the genomic RNA, regulates the synthesis of viral RNA, and plays a key role in participating in viral replication and transcription. Based on the in-depth study of the performance of the PEDV N protein, the applicant's research team clarified the infection regulation mechanism of PEDV and innovatively proposed to use the PEDVN protein as the core of the IPEC-J2 cell line to provide a new idea for solving the problem. In the existing publicly reported research results, there is no clear record of using the characteristics of the nucleocapsid (N) protein to increase the susceptibility of IPEC-J2 cells to PEDV.
[0022] To this end, the applicant conducted in-depth research and repeated verification on PEDV N, and finally confirmed that the nucleocapsid protein is the key structural protein affecting PEDV replication. On this basis, the applicant further proposed the following IPEC-J2 cell line design: based on the PEDV N protein sequence, a truncated fragment DNA for targeting the virus was designed, and a fluorescent tag protein (CopGFP) and a puromycin resistance gene (Puro) were added to the constructed CD513B-1 lentiviral vector. The former facilitates direct observation of infection efficiency by fluorescence, and the latter is used to screen and purify cells.
[0023] The present invention directly infects the cell line with PEDV according to a common infection method, and then harvests proteins and total RNA by lysing the cells, detects the expression of viral PEDV N protein in the cells to reflect the replication level of the virus, and further reflects the infection of the virus by immunofluorescence and virus titer determination. In the specific operation, the R58-WT-IPEC cell line is finally obtained by inserting the target fragment, packaging the lentivirus, infecting the target cells, and screening with puromycin. Through comparative verification of PEDV infection, the results show that the cell line can significantly enhance the replication of PEDV.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] 1. Through in-depth research, the present invention reveals the key role of PEDV N protein in the viral replication stage, and proposes to use it as a target for regulating PEDV infection.
[0026] 2. The present invention provides an IPEC-J2 cell line that is beneficial to virus replication based on a new target; compared with the wild-type IPEC-J2 cell line, the cell line has a significant promoting effect, can solve the problem of poor proliferation of PEDV in the IPEC-J2 cell line, and further enhances the possibility of using the IPEC-J2 cell line in virus isolation and propagation.
[0027] 3. The cell line prepared by the present invention can maintain its characteristics stably for a long time, which helps to gain a deeper understanding of the regulatory mechanism of PEDV N in coronavirus infection, is very important for revealing the infection and spread of the virus, and can be used for virus isolation, propagation and the study of new cell models in the regulatory mechanism of PEDV infection. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 is the spectrum of R58-WT-CD513B;
[0029] Figure 2 This is the morphological diagram of the R58-WT-IPEC cell line;
[0030] Figure 3 This is the fluorescence image of the R58-WT-IPEC cell line;
[0031] Figure 4 Analysis of PEDV infection in R58-WT-IPEC cells. DETAILED DESCRIPTION
[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments and the accompanying drawings, but they should not be understood as limiting the scope of protection of the present invention. The present invention is not limited to the following embodiments, and the test methods not described in detail in the embodiments are all conventional methods in the art.
[0033] The reagents used in the present invention are all commonly used reagents in biological laboratories. The wild-type IPEC-J2 cells were isolated and preserved from the intestines of pigs raised in a farm in Zhejiang Province by the applicant's research team.
[0034] Example 1 Construction of IPEC-J2 cell line carrying PEDV N protein truncated fragment
[0035] 1) Construction of lentiviral vector targeting PEDV N protein truncated fragment
[0036] The PEDV N protein sequence (GenBank: MK409659.1) was obtained from the NCBI database, and a DNA sequence targeting the truncated fragment of the protein sequence was designed, as shown in SEQ ID NO: 1, and Beijing Qingke Biotechnology Co., Ltd. was commissioned to synthesize it, namely R58-WT-fra. Subsequently, the synthesized DNA was inserted into the CD513B-1 vector by homologous recombination and named R58-WT-CD513B. The map is shown in Figure 1 The ligation product was transformed into DH5α and evenly spread on an LB plate containing ampicillin. The single colony obtained by antibiotic screening was sent to a sequencing company for sequencing after PCR identification. The expected positive colony was expanded and cultured, and the plasmid was extracted to obtain a lentiviral recombinant plasmid targeting the truncated fragment of the PEDV N protein for later use.
[0037] 2) Lentiviral packaging
[0038] Take the 293T cells in the best state and spread them in a six-well plate. When the cell density reaches 70%, wash and change the medium in advance, and add 1.8mL of DMEM medium containing 10% fetal bovine serum without antibiotics. Then mix 8μL HighGene Transfection reagent transfection reagent with 4μg of lentiviral recombinant plasmid, packaging plasmid pMD2.G, pMDlgpRRE, and pRSV-Rev in 200μL opti-MEM medium, incubate at room temperature for 15min, and then drop into the prepared 293T cells and culture in a 37℃, 5% CO2 incubator. Since the recombinant plasmid is inserted with a CopGFP tag sequence, it can express green fluorescent protein, so the transfection efficiency can be judged by observing the fluorescence. After 36h of transfection, the fluorescence rate reaches about 80%, collect the supernatant of 293T cells containing the virus, add 2mL of the above culture medium, and collect the supernatant of 293T cells containing the virus again after 48h, which is the virus liquid. Filter through a 0.22μm filter and package, and store at 4℃ for one week, or at -80℃ for long-term storage.
[0039] 3) Infection of IPEC-J2 cells with lentivirus
[0040] Wild-type IPEC-J2 cells were plated in a six-well plate. When the cell density reached about 30%, 1 mL of virus solution was added to the well after washing. The cell status was observed 4 hours after infection. If the cell status was good, it was extended to 6 hours. Then 1 mL of complete medium was added and cultured overnight. After 24 hours, the fluorescence was observed, the culture medium was discarded and washed, and 1 mL of virus solution was added to the well. After 48 hours, the cell status was observed 4 hours after infection. If the cell status was good, it was extended to 6 hours. Then 1 mL of complete medium was added and cultured overnight. After 48 hours, the fluorescence was observed again and the above operation was repeated. The fluorescence was continued to be observed at 72 hours. When the fluorescence rate reached 60% to 70%, puromycin could be added for drug screening.
[0041] 4) Puromycin screening of stable cell lines
[0042] The IPEC-J2 cells co-expressing CopGFP green fluorescent protein and PEDV N truncated protein were passaged at a ratio of 1:4, and 2.5 μg / mL of puromycin was added for screening and culture; on the second day of passage, the medium was changed and antibiotic-free complete medium was added, and cell fluorescence was observed; when the IPEC-J2 cell density reached about 80%, the cells were passaged at a ratio of 1:4, and 5 μg / mL of puromycin was added for screening and culture; on the second day of passage, the medium was changed and antibiotic-free complete medium was added, and cell fluorescence was observed; the above steps were repeated, and the puromycin screening concentration was gradually increased until the cell line was stable and could be used for subsequent experiments. The cell line was named R58-WT-IPEC. Figure 2 It can be seen that this cell line has the same morphology as IPEC-J2 cells.
[0043] The sequence of the puromycin resistance gene (Puro) is shown in SEQ ID NO: 3. R58-WT-IPEC cells containing the puromycin resistance gene can grow in an environment treated with puromycin, while wild-type IPEC cannot survive. Based on this characteristic, it is an effective means to screen stable cells. After testing, R58-WT-IPEC cells containing the puromycin resistance gene can grow normally in a culture environment of 10 μg / mL puromycin and have strong EGFP fluorescence, indicating that the stable cell line was successfully constructed ( Figure 3 ).
[0044] In addition, the applicant submitted the R58-WT-IPEC cell line to the China Center for Type Culture Collection for preservation on October 24, 2024, and the survival status is alive; its preservation number is CCTCC NO: C202425; the preservation address is Wuhan, China (Wuhan University), the postal code is 430072, and the contact number is (027) 68754833.
[0045] Example 2 Experimental verification that the R58-WT-IPEC cell line is beneficial for PEDV infection
[0046] Experimental methods: Wild-type IPEC-J2 cells and R58-WT-IPEC cells were seeded into six-well plates, grown to a density of 60%, infected with PEDV at MOI = 1 for 24 hours, and three sets of parallel wells were made for qPCR, Western blot, IFA and virus titer determination. Western blot detected the protein level of PEDV-N. Grayscale analysis determined the relative expression of PEDV-N and β-actin. The experiment was repeated three times and displayed as means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001. qPCR detected the mRNA level of PEDV-N. IFA and virus titer intuitively showed the infection difference of PEDV in the two IPEC-J2 cells.
[0047] The test results showed that after PEDV infected R58-WT-IPEC cells, the expression of PEDV N was significantly enhanced ( Figure 4 A, B and C), plus its virus titer ( Figure 4 D), the number of PEDV-N positive cells increased significantly ( Figure 4 E and F), indicating that the cell line promotes PEDV infection. Therefore, it is proved that the R58-WT-IPEC cell line provided by the present invention can significantly promote the PEDV replication process, and can be further used to enhance the susceptibility of IPEC to PEDV, thereby realizing the application in virus isolation and passage.
[0048] The protection scope of the present invention is not limited to the above-mentioned specific embodiments; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments rather than to limit the protection scope of the present invention. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the technical personnel in the field of the present invention's mastery of the prior art and the records of the present invention, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to implement the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
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Claims
1. An IPEC-J2 cell line stably expressing a truncated fragment of PED virus nucleocapsid protein, characterized in that: The cell line is deposited in China Center for Type Culture Collection, the deposited name is porcine small intestinal epithelial cell line R58-WT-IPEC, the Latin name is Sus scrofa, and the deposited number is CCTCC NO: C202425.
2. The IPEC-J2 cell line according to claim 1, characterized in that The cell line is a stably transfected cell line targeting the PEDV replication stage, and can stably express the target fragment sequence shown in SEQ ID NO: 1 in the PEDV nucleocapsid protein.
3. The IPEC-J2 cell line according to claim 1, characterized in that The cell line has both a fluorescent label protein and a puromycin resistance gene, the sequence of the former is shown in SEQ ID NO:2, and the sequence of the latter is shown in SEQ ID NO:
3.
4. The method for constructing the IPEC-J2 cell line according to claim 1, characterized in that: The following steps are involved: (1) Vector construction: The target truncated fragment of PEDV N protein was inserted into the CD513B-1 vector by homologous recombination; After transformation and sequencing identification, the lentiviral recombinant plasmid was obtained; (2) Lentiviral packaging: The lentiviral recombinant plasmid and packaging plasmids pMD2.G, pMDlgpRRE, and pRSV-Rev were co-transfected into 293T cells to obtain viral fluid; (3) Infecting IPEC-J2 cells with lentivirus: Increasing the amount of lentivirus infection in IPEC-J2 cells by repeated infection; (4) Screening of stable cell lines: Positive cell lines were screened by adding puromycin and gradually increasing the concentration to obtain the IPEC-J2 cell line carrying the truncated fragment of the PEDV N protein.
5. The method according to claim 4, characterized in that The sequence of the CD513B-1 vector is shown in SEQ ID NO:
4.
6. The method according to claim 4, characterized in that The IPEC-J2 cell line carrying the truncated fragment of the PEDV N protein can grow normally in a culture environment of 10 μg / mL puromycin.
7. Use of the IPEC-J2 cell line according to claim 1 in promoting PEDV replication.
8. Use of the IPEC-J2 cells according to claim 1 in the preparation of porcine epidemic diarrhea virus vaccines and drugs.