Application of MIDN gene in prevention and treatment of porcine reproductive and respiratory syndrome virus

Abstract

The invention discloses application of an MIDN gene in prevention and treatment of porcine reproductive and respiratory syndrome virus, and belongs to the technical field of veterinary biological products. The research finds that the MIDN gene participates in the PRRSV infection process for the first time, PRRSV infection can be remarkably inhibited through overexpression of the MIDN gene, and the MIDN gene can be used for preventing and treating the PRRSV; by knocking out the MIDN gene in an animal cell line, PRRSV infection of the cell line can be promoted. The porcine reproductive and respiratory syndrome virus super-susceptible cell line provided by the invention has super-strong infection ability to different types of PRRSV, and can increase the virus infection titer and shorten the infection time; the method can be used for rapid high-titer preparation of PRRSV, and provides a susceptible cell line for efficient production of PRRSV vaccines.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to an application of the MIDN gene in the prevention and treatment of porcine reproductive and respiratory syndrome virus. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is an acute infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The disease's primary clinical features include reproductive failure in sows and respiratory distress in pigs of all ages, causing significant economic losses to the swine industry.

[0003] Currently, live attenuated vaccines are the primary clinical approach for the prevention and treatment of PRRSV. These vaccines require inoculating animal cells with a standard virus strain used in vaccine production. After a suitable period of incubation, the cell culture is harvested. The isolated and purified virus then serves as the direct or indirect raw material for viral vaccine production. Therefore, establishing passage cells and obtaining high-titer, immunogenic adapted strains is a key technology in vaccine production.

[0004] MIDN protein (Midnolin) is a nuclear protein that has attracted much attention in recent years. Its functions involve multiple fields such as protein degradation regulation, neural development and disease mechanisms. The MIDN-proteasome pathway is a new protein degradation mechanism that bypasses the traditional ubiquitination system and directly mediates the degradation of key transcription factors in the nucleus. For example, in the nervous system, MIDN regulates synaptic plasticity and memory formation by degrading short-lived proteins (such as Fos and EGR1) expressed by immediate early genes (IEGs). In the immune system, MIDN may affect the function of immune cells by degrading transcription factors such as IRF4. Studies have found that MIDN is highly expressed in PC12 cells (neuronal model), and its knockout significantly inhibits neurite outgrowth, suggesting its key role in neuronal differentiation. However, there are currently no reports on the role of MIDN protein in PRRSV infection. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide an application of MIDN gene in the prevention and treatment of porcine reproductive and respiratory syndrome virus.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides the use of an overexpressed MIDN gene in the preparation of a drug for inhibiting PRRSV infection;

[0008] The MIDN gene is a DNA molecule as shown in the following i) or ii):

[0009] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0010] ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO. 2 except i).

[0011] In the above application, the PRRSV is preferably HP-PRRSV.

[0012] The present invention has found that the MIDN gene is involved in the infection process of PRRSV, and that overexpression of the MIDN gene can significantly inhibit PRRSV infection.

[0013] The second aspect of the present invention provides the use of MIDN protein in the preparation of a drug for inhibiting PRRSV infection; the amino acid sequence of the MIDN protein is shown in SEQ ID NO.2.

[0014] Genes direct protein synthesis through transcription and translation, so proteins are the executors of gene function. MIDN protein can inhibit PRRSV infection, and therefore, MIDN protein can be used as an active ingredient in the preparation of drugs that inhibit PRRSV infection.

[0015] The third aspect of the present invention provides the use of a recombinant expression vector or genetically engineered bacteria containing the MIDN gene in the preparation of a drug for inhibiting PRRSV infection.

[0016] The fourth aspect of the present invention provides the use of knocking out the MIDN gene in constructing a porcine reproductive and respiratory syndrome virus super-susceptible cell line.

[0017] The present invention has found that knocking out the MIDN gene in Marc-145 cells can enhance the infection ability of PRRSV to Marc-145 cells, increase the virus infection titer, and shorten the infection time; it can be used for the rapid high-titer preparation of PRRSV, and at the same time provide a susceptible cell line for the efficient production of PRRSV vaccine.

[0018] A fifth aspect of the present invention provides a porcine reproductive and respiratory syndrome virus hypersusceptible cell line constructed by knocking out the MIDN gene in animal cells.

[0019] Preferably, the animal cells are Marc-145 cells.

[0020] Preferably, MIDN gene knockout is performed by the following method:

[0021] Target 1 and target 2 were used as double knockout target sequences of the MIDN gene, and the MIDN gene was knocked out in animal cells using CRISPR-Case9 technology;

[0022] The sense strand sequence of target 1 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.4; the sense strand sequence of target 2 is shown in SEQ ID NO.5, and the antisense strand sequence is shown in SEQ ID NO.6. The details are as follows:

[0023] Target 1 positive chain: CACCGCGTCTCCGTGAGACTCTCGAGGG; (SEQ ID NO.3)

[0024] Target 1 antisense strand: AAACCCCTCGAGAGTCTCACGGAGACGC. (SEQ ID NO. 4)

[0025] Target 2 positive chain: CACCGAAAGCTCTCGATGACGGCGCCGG; (SEQ ID NO.5)

[0026] Target 2 antisense strand: AAACCCGGCGCCGTCATCGAGAGCTTTC. (SEQ ID NO.6)

[0027] The dual-target knockout method of the present invention can knock out the MIDN gene in animal cells without off-target effects; therefore, the constructed porcine reproductive and respiratory syndrome virus super-susceptible cell line is actually a MIDN knockdown cell line, and it is named MIDN-KO, and knocking down the MIDN gene does not change the cell growth characteristics.

[0028] The sixth aspect of the present invention provides the use of the porcine reproductive and respiratory syndrome virus hypersusceptible cell line in the following (1) or (2):

[0029] (1) preparing a high titer PRRSV virus solution;

[0030] (2) Production of PRRSV vaccine.

[0031] In the above application, the PRRSV is HP-PRRSV, LP-PRRSV, NADC30 or NADC30-like.

[0032] The cell line constructed by the present invention can support the rapid propagation of different types of PRRSV, increase the virus infection titer and shorten the infection time; can be used for the rapid high-titer preparation of PRRSV, and at the same time provide an efficient cell line for the production of PRRSV vaccine.

[0033] Beneficial effects of the present invention:

[0034] (1) The present invention first discovered that the MIDN gene is involved in the infection process of PRRSV. By overexpressing the MIDN gene, PRRSV infection can be significantly inhibited, and it can be used for the prevention and treatment of HP-PRRSV. By knocking out the MIDN gene in animal cell lines, PRRSV infection of cell lines can be promoted.

[0035] (2) Based on the newly discovered function of the MIDN gene, the present invention knocked out the MIDN gene in Marc-145 cells and constructed a porcine reproductive and respiratory syndrome virus supersusceptible cell line. The porcine reproductive and respiratory syndrome virus supersusceptible cell line of the present invention has a strong infection ability for different types of PRRSV (HP-PRRSV, LP-PRRSV, NADC30 and NADC30-like), can increase the virus infection titer and shorten the infection time; can be used for the rapid high-titer preparation of PRRSV, and at the same time provide a susceptible cell line for the efficient production of PRRSV vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 :The effect of HP-PRRSV infection on MIDN expression;

[0037] Western blot detection results (A) and Real-time PCR detection results (B) of the effect of TA-12 infection on MIDN mRNA levels in Marc-145 cells; Western blot detection results (C) and Real-time PCR detection results (D) of the effect of TA-12 infection on MIDN mRNA levels in PAM cells.

[0038] Figure 2 :The effect of MIDN on TA-12 infection;

[0039] Western blot (A) detection of PRRSV N protein after overexpression of MIDN in Marc-145 cells and Real-time PCR (B) detection results of PRRSV N gene by MIDN.

[0040] Figure 3 :Effects of MIDN knockdown on TA-12 infection;

[0041] Western blot results of MIDN expression in MIDN knockdown cell lines and its effect on TA-12 infection (A), Real-time PCR detection results of MIDN gene expression in MIDN knockdown cell lines (B), CCK-8 results of the effect of MIDN knockdown cell lines on cell viability (C), Real-time PCR detection results of the effect of MIDN knockdown in MIDN knockdown cell lines on TA-12 infection (D), and viral titer detection results (E).

[0042] Figure 4 :The effect of MIDN knockdown on MIDN knockdown cell lines on TA-01, TA-02 and CH-1R infection;

[0043] Western blot (A) test results, Real-time PCR (B, C, D) test results, and viral titer (E, F, G) test results of the effect of MIDN knockdown on TA-01, TA-02, and CH-1R infection. DETAILED DESCRIPTION

[0044] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0045] As mentioned above, porcine reproductive and respiratory syndrome (PRRS) caused by PRRSV has caused huge economic losses to the pig industry. The main technical difficulties faced in the prevention and control of PRRSV are: (1) the lack of susceptible cell lines that can produce PRRSV vaccines in large quantities; (2) due to the rapid onset and spread of HP-PRRSV, the vaccine immunity is not ideal, making it difficult to prevent and control HP-PRRSV.

[0046] In view of this, the present invention conducted an in-depth study on the infection process of PRRSV and found that the MIDN gene is involved in the infection process of PRRSV. The nucleotide sequence of the MIDN gene is shown in SEQ ID NO. 1, which is as follows:

[0047] atggagccgcagcccggcggcgcccggagctgccggcgcggggcccccggcggcgcctgcgagctgggcccggcggccgaggc

[0048] ggcgcccatgagcctggccatccacagcaccacgggcactcgctacgacctggccgtgccgcccgatgagacggtggaggggctgcg

[0049] caagcggttgtctcagcgcctgaaagtgcccaaggagcgcctggcgcttctccacaaagacacccggctcagttcggggaagctgcag

[0050] gagttcggcgtgggtgatggcagcaagctgaccttggtacccaccgtggaagcgggcctcatgtctcaggcctcaaggccggagcagtc

[0051] tgtgatgcaagccctcgagagtctcacggagacgcaggtcagtgacttcctgtcgggccgctcgccactgacactggccttgcgtgtggg

[0052] cgaccacatgatgttcgtacagctgcagctcgcggcccagcacgctccactgcaacaccgccatgtgctggctgccgccgccgccgccg

[0053] ccgctgctgcacggggggacccaagcatagcctcccccgtgtcctcaccctgccggcccatgtccagtgctgcccgagtccccccggtg

[0054] cccacgagcccctcccctgcgtctccctcgcccatcacagccggctccttccggtcccacgcagcctccaccacctgcccagagcagat

[0055] ggactgctcccccacggccagcagcagtgccagtcctggtgccagcaccgcgtctaccccaggggccagccctgccccccgctcccg

[0056] aaaacccggcgccgtcatcgagagctttgtgaatcatgccccgggggtcttctcagggaccttctctggcacgctacaccccaactgccaa

[0057] gacagcagcgggcggccacggcgtgacatcggcaccatcctgcagatcctcaatgacctcctgagcgccacccggcactaccagggc

[0058] atgcccccctcgctggcccagctccgctgccacgcccagtgctccccggcttcgccggcccccgacctggcccccagaactacctcctg

[0059] cgagaagctcacggctgcccccgcagcctccctgctgcagagccagatccgcatgtgcaagcctccgggggaccggcttcggcagac

[0060] ggaaaaccgcgccacacgctgcaaggtggaacggctgcagctgctcctgcagcagaaacggctgcgtagaaaggcccggcgggacg

[0061] cgcggggtccgtaccactggtcacccagccgcaaggccggccgaagcgacagcagtagcagcgggggcggtggcagctccagcgaggcctccggcctgggcctcgacttcgaggactccgtgtggaagccagaagtcaaccctgacatcaagtcagagttcgtggtggcttag。

[0062] The amino acid sequence of the MIDN protein encoded by the MIDN gene is shown in SEQ ID NO.2, as follows:

[0063] MEPQPGGARSCRRGAPGGACELGPAAEAAPMSLAIHSTTGTRYDLAVPPDETVEGLRKRLSQRLKVPKERLALLHKDTRLSSGKLQEFGVGDGSKLTLVPTVEAGLMSQASRPEQSV MQALESLTETQVSDFLSGRSPLTLALRVGDHMMFVQLQLAAQHAPLQHRHVLAAAAAAAAAARGDPSIASPVSSPCRPMSSAARVPPVPTSPSPASPSPITAGSFRSHAASTTCPEQ MDCSPTASSSASPGASTASTPGASPAPRSRKPGAVIESFVNHAPGVFSGTFSGTLHPNCQDSSGRPRRDIGTILQILNDLLSATRHYQGMPPSLAQLRCHAQCSPASPAPDLAPRTT SCEKLTAAPAASLLQSQIRMCKPPGDRLRQTENRATRCKVERLQLLLQQKRLRRKARRDARGPYHWSPSRKAGRSDSSSSGGGGSSSEASGLGLDFEDSVWKPEVNPDIKSEFVVA.

[0064] The present invention overexpresses the MIDN gene in Marc-145 and then infects HP-PRRSV, and finds that the overexpression of the MIDN gene can inhibit the infection of HP-PRRSV.

[0065] The present invention further uses CRISPR-Cas9 technology to knock down MIDN in Marc-145 cells to construct a MIDN-KO cell line. The CRISPR-Cas9 system is a widely used genome editing tool that comes from the adaptive immune system of bacteria. The CRISPR-Cas9 system includes: a Cas9 enzyme and a guide RNA. The function of the guide RNA is to guide Cas9 to cut at a specific site in the genome. So far, the CRISPR-Cas9 system has two main applications: gene knockout and gene knock-in. For gene knockout, once the double-strand break reaction (DSB) of DNA is induced by Cas9 cutting, the cell will initiate the NHEJ DNA repair method, which will cause the deletion and insertion of DNA, but the disadvantage of this method is that it is easy to cause off-target effects, resulting in incomplete knockout of the target gene or no knockout. To achieve effective knockout of MIDN, the present invention optimizes the CRISPR-Cas9 system and uses dual target sites for gene knockout in the two exon regions of the MIDN gene. By constructing a vector inserting the dual target sites and transfecting it into Marc-145 cells, the present invention can achieve effective knockout of MIDN in Marc-145 cells and prevent off-target effects, thereby ensuring the stability of MIDN-KO and having no effect on cell activity and growth characteristics.

[0066] The MIDN-KO constructed in the present invention has a super strong infection ability to different types of PRRSV, can increase the virus infection titer and shorten the infection time; and can be used for the rapid high-titer preparation of PRRSV.

[0067] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific embodiments.

[0068] The test materials used in the embodiments of the present invention are all conventional test materials in the field and can be purchased through commercial channels. Experimental methods without detailed conditions are carried out in accordance with conventional test methods or in accordance with the operating instructions recommended by the supplier. Among them: the PRRSV strains used in the present invention, the HP-PRRSV type is represented by the TA-12 strain, the LP-PRRSV type is represented by the CH-1R strain, the NADC30-like type is represented by the TA-01 strain, and the NADC30 recombinant type is represented by the TA-02 strain. The above strains are all known strains in the prior art, and the public can obtain the above strains from the applicant within 20 years from the date of application for use in repeating the present invention.

[0069] If specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are generally followed, such as those in J. Sambrook et al., ed., Molecular Cloning Experiment Manual (3rd edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Manual, Science Press, 2001; or the conditions recommended by the manufacturer.

[0070] Example 1: MIDN is involved in PRRSV infection

[0071] We first verified the effect of TA-12 infection on MIDN expression in Marc-145 and PAM cells. TA-12 was inoculated at an MOI of 0.1 on monolayers of Marc-145 and PAM cells. One hour later, the culture medium was replaced with 2% fetal bovine serum. The cells were washed with pre-cooled PBS at 0, 12, 24, and 36 hours after infection. All cells were blown off with a pipette and transferred to a new centrifuge tube. The tube was centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and the precipitate was retained. The cells were lysed with cell lysis buffer to collect proteins for Western blot verification. At the same time, cellular RNA was extracted and reverse transcribed for fluorescence quantitative PCR verification (see Table 1 for primers used for fluorescence quantitative PCR). Western blot results showed that TA-12 infection upregulated the protein level of MIDN (see Table 1). Figure 1 A, C); Real-time PCR results showed that TA-12 infection also upregulated the mRNA level of MIDN (see Figure 1 B, D).

[0072] Table 1: Primers used for fluorescence quantification

[0073]

[0074] Note: M in MIDN(M) stands for monkey, and this assay detects the MIDN gene in Marc-145 cells. S in MIDN(S) stands for swine, and this assay detects the MIDN gene in PAM cells.

[0075] Example 2: MIDN inhibits PRRSV infection

[0076] To further investigate the role of MIDN in PRRSV infection, we overexpressed MIDN in Marc-145 cells and then infected TA-12 cells to verify the effect of MIDN on PRRSV infection.

[0077] The MIDN gene was constructed into the eukaryotic expression vector pCMV-myc by homologous recombination. After plating Marc-145 cells, transfection was performed when the cell density reached 80%. 3 μg of plasmid pCMV-myc-MIDN and its empty vector pCMV-myc were transfected into Marc-145 cells using ExFect Transfection Reagent (Vazyme, NanJing, CHN) according to the manufacturer's instructions. 24 hours after transfection, TA-12 cells were infected at an MOI of 0.01. At different time points (0 h, 12 h, 24 h, and 36 h) after TA-12 infection, cells were harvested for Western blot and fluorescence quantitative PCR verification. Western blot and fluorescence quantitative PCR results showed that overexpression of MIDN significantly inhibited HP-PRRSV infection ( Figure 2 ).

[0078] Example 3: Construction of MIDN knockout cell line

[0079] The present invention constructs MIDN knockout cell line for next step verification, and the CRISPR-Cas9 vector system carrying two guide RNA expression cassettes is used to construct a recombinant plasmid containing target 1 and target 2 (target 1 and target 2 sequences are shown in SEQ ID NO.3-SEQ ID NO.6 in the sequence table). First, the corresponding primers are synthesized according to the target 1 and target 2 sequences, and after the forward primer and reverse primer are mixed, the primers are phosphorylated and annealed in a PCR instrument for standby use. Then, the PX459M and EZ-GuideXH vectors are further digested with BbsI enzyme, and the digested products are purified by gel recovery. Next, target 1 and PX459M vectors and target 2 and EZ-GuideXH vectors are connected to the transformation experiment and positive clones and sequencing verification are identified. Next, we digest target 2 from the EZ-GuideXH vector and connect it to the PX459M vector containing target 1 to construct a vector (recombinant CRISPR / Cas9 KO plasmid) inserted into the dual target point and further identify positive clones and sequencing verification.

[0080] The identified recombinant CRISPR / Cas9 KO plasmid was transfected into Marc145 cells, and then the medium containing 10% FBS and 10 μg / mL puromycin antibiotics was replaced with ordinary culture medium containing 10% FBS and 1% double antibody approximately every 2-3 days, and the successfully transfected positive cells that did not die were screened for at least 3-5 days. Western blot ( Figure 3 A) and fluorescence quantification ( Figure 3B) Confirm the knockout effect. The results showed that MIDN in this cell line had a significant knockdown effect, and it was named MIDN-KO. The blank vector was also treated with the same procedure, and the corresponding cell line was named VEC. At the same time, CCK-8 assay ( Figure 3 C) The effect of MIDN knockdown on cell viability was verified, and the results showed that MIDN knockdown had no adverse effect on cell viability.

[0081] HP-PRRSV (TA-12) was infected on MIDN-KO and VEC cells, and the effect on HP-PRRSV infection was detected by Western blot. Western blot results showed that HP-PRRSV infection in the knockdown group increased significantly at 24 and 36 h after infection ( Figure 3 A). Similarly, cells were collected to extract RNA and the cell supernatant was used for fluorescence quantification and virus titration. Real-time PCR results showed that MIDN knockdown could significantly increase the viral copy number of TA-12 ( Figure 3 D), the viral titer results showed that MIDN knockdown could increase the viral titer of TA-12 ( Figure 3 E).

[0082] Example 4: Increased infection of different PRRSV strains on MIDN-KO cell lines

[0083] TA-01, TA-02, and CH-1R were inoculated at a dose of 0.5 MOI on the monolayer MIDN-KO cells constructed in Example 3, and the cells were harvested at different times after infection for Western blot verification, and cellular RNA was extracted and reverse transcribed for real-time verification.

[0084] The results showed that N protein was significantly increased in TA-01, TA-02 and CH-1R cells infected with MIDN-KO compared with VEC cell lines ( Figure 4 A), further fluorescence quantitative detection revealed that after MIDN knockdown, the copy numbers of TA-01, TA-02, and CH-1R increased significantly at 12, 24, and 36 h after infection ( Figure 4 B, C, D). The results of virus titer determination also showed that knockdown of MIDN could increase the virus titers of three different strains (see Figure 4 E, F, G).

[0085] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Application of overexpressed MIDN gene in the preparation of drugs for inhibiting PRRSV infection; The MIDN gene is a DNA molecule as shown in i) or ii) below: i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The use according to claim 1, characterized in that: The PRRSV is HP-PRRSV.

3. The use of MIDN protein in the preparation of a drug for inhibiting PRRSV infection, characterized in that: The amino acid sequence of the MIDN protein is shown in SEQ ID NO.

2.

4. Use of a recombinant expression vector or genetically engineered bacteria containing the MIDN gene in the preparation of a drug for inhibiting PRRSV infection.

5. Application of knocking out MIDN gene in constructing porcine reproductive and respiratory syndrome virus super-susceptible cell line, wherein the MIDN gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

6. A porcine reproductive and respiratory syndrome virus supersusceptible cell line, characterized in that: It is constructed by knocking out the MIDN gene in animal cells.

7. The porcine reproductive and respiratory syndrome virus super-susceptible cell line according to claim 6, characterized in that: The animal cells are Marc-145 cells.

8. The porcine reproductive and respiratory syndrome virus super-susceptible cell line according to claim 6, characterized in that: MIDN gene knockout was performed using the following method: Using target 1 and target 2 as double knockout target sequences of the MIDN gene, the MIDN gene in animal cells was knocked out using CRISPR-Case9 technology; The sense strand sequence of the target 1 is shown in SEQ ID NO.3, and the antisense strand sequence is shown in SEQ ID NO.4; the sense strand sequence of the target 2 is shown in SEQ ID NO.5, and the antisense strand sequence is shown in SEQ ID NO.

6.

9. Use of the porcine reproductive and respiratory syndrome virus supersusceptible cell line according to any one of claims 6 to 8 in the following (1) or (2): (1) preparing a high titer PRRSV virus solution; (2) Production of PRRSV vaccine.

10. The use according to claim 9, characterized in that: The PRRSV is HP-PRRSV, LP-PRRSV, NADC30 or NADC30-like.

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