Application of MARCH5 in identifying susceptibility of pigs to porcine reproductive and respiratory syndrome virus and preparing drugs

Abstract

The present invention discloses the application of MARCH5 in identifying the susceptibility of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) and in the preparation of drugs. The research of the present invention shows that the expression level of MARCH5 in pigs is closely related to PRRSV infection. After PRRSV infects porcine alveolar macrophages (PAMs) cells, the levels of MARCH5 mRNA and protein increase significantly. Interference with MARCH5 promotes PRRSV replication, while overexpression of MARCH5 inhibits virus replication. When the expression level of MARCH5 in pigs is relatively high, pigs are resistant to PRRSV, and vice versa, they are susceptible. Therefore, MARCH5 can be used as a criterion for determining the susceptibility or resistance to PRRSV, laying a solid foundation for the identification of the susceptibility of pigs to PRRSV and the screening, identification and breeding of PRRSV-resistant pigs, which is beneficial to the breeding of excellent pig strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to the application of MARCH5 in identifying the susceptibility of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) and in the preparation of drugs. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as porcine blue ear disease, is caused by Porcine reproductive and respiratory syndrome virus (PRRSV). As early as 1992, the World Organization for Animal Health (OIE) listed this disease as a Class B infectious disease. Currently, PRRS is one of the most important infectious diseases in the pig industry, almost distributed in all pig-raising countries, causing huge economic losses to the global pig industry. Moreover, PRRSV can also trigger porcine high fever syndrome (PHFS), which has caused huge economic losses to the pig industry. This disease is mutated from PRRSV and has characteristics such as high fever, high pathogenicity, and high mortality. Therefore, the prevention and control of PRRS is a difficult problem in China and even in the world.

[0003] Currently, the difficulties in preventing and controlling PRRS are mainly manifested in the following aspects: (1) It is a macrophage-tropic and immunosuppressive disease. PRRSV mainly infects immune cells - porcine alveolar macrophages (PAMs), destroying the immune system and causing immunosuppression. Therefore, it is known as "AIDS in humans and blue ear in pigs"; (2) The pathogen is extremely easy to mutate. PRRSV is an RNA virus and mutates very quickly. The use of live attenuated vaccines in the field is an important reason for promoting virus mutation. There are literature reports of isolating PRRSV virulent strains with a high degree of genomic homology to the vaccine strain from pig farms, and the virulence has increased. It is analyzed that it may be the reversion of the vaccine virus to a virulent form or recombination with field strains; (3) The vaccines do not have cross-protective ability. Currently, the commercial vaccines against PRRSV on the market have weak cross-protective ability against different strains; (4) Antibody-dependent enhancement (ADE). The infection of PRRSV will stimulate the body to produce antibodies, but medium and low-titer antibodies not only cannot neutralize the virus, but instead promote the proliferation of the virus; (5) Persistent virus infection. After PRRSV infection, viremia can be detected in pigs for a long time, and the duration of PRRSV in the body can be up to 5 months; (6) Co-infection. Currently, co-infection of blue ear with other diseases is common in clinical practice, especially the co-infection of PRRSV with porcine circovirus, Haemophilus parasuis, and swine plague, making the prevention and control of PRRS even more difficult.

[0004] In summary, the vaccines developed from the perspective of the pathogen (PRRSV) have the following problems: slow generation of neutralizing antibodies, poor cross-protection against different strains, long virus-carrying time of the vaccine virus, and recombination with field strains to produce recombinant viruses with stronger pathogenicity. Therefore, it is necessary to conduct research from the genetic perspective of the host (pig) to improve the host's disease resistance to PRRS at the genetic level. Since the genetic background is closely related to biological disease resistance, the work of screening PRRS-resistant pigs based on genetic means has incomparable advantages for the prevention and control of this disease. The key lies in finding genes related to PRRSV host resistance or susceptibility, which can not only study the infection mechanism of PRRS but also provide new methods for the treatment and prevention of PRRS. For example, Chinese Patent CN106191320A discloses the application of the HPSE gene in screening PRRS-resistant pigs. By exploring PRRSV host disease-resistant / susceptible genes, new varieties (lines) of disease-resistant (PRRS) pigs can be cultivated and screened, and exploring more related genes is of great significance for molecular-level resistance to PRRS. Summary of the Invention

[0005] The present invention aims to provide the application of MARCH5 in identifying the susceptibility of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) and in the preparation of drugs. Specifically:

[0006] The first object of the present invention is to provide the application of a detection reagent for the MARCH5 gene or protein.

[0007] The second object of the present invention is to provide the application of the MARCH5 gene in cultivating pig breeds resistant to porcine reproductive and respiratory syndrome.

[0008] The third object of the present invention is to provide a method for screening pigs resistant to porcine reproductive and respiratory syndrome.

[0009] The fourth object of the present invention is to provide a preparation for improving the resistance of pigs to porcine reproductive and respiratory syndrome virus.

[0010] The fifth object of the present invention is to provide a method for cultivating pigs resistant to porcine reproductive and respiratory syndrome.

[0011] The above objects of the present invention are achieved by the following technical solutions:

[0012] The research of the present invention shows that after PRRSV infects PAMs cells, it promotes the expression of membrane-associated ring-CH-type finger 5 (MARCH5). As the infection time increases, the expression level of MARCH5 increases significantly. While the interference of MARCH5 promotes the replication of PRRSV, and the overexpression of MARCH5 inhibits the replication of PRRSV, indicating that the expression level of MARCH5 is closely related to the replication of PRRSV. At the same time, the research shows that high expression of MARCH5 in pigs is resistant to porcine reproductive and respiratory syndrome virus (PRRSV), and vice versa, it is susceptible. It has good clinical application value for the cultivation of PRRSV-resistant pigs.

[0013] The MARCH5 mRNA sequence number of the present invention research: XM_001927572, and the MARCH5 protein sequence number: XP_001927607.

[0014] Therefore, the present invention provides:

[0015] The application of a detection reagent for MARCH5 gene or protein in the preparation of products for detecting and diagnosing porcine reproductive and respiratory syndrome (PRRS).

[0016] The application of a detection reagent for MARCH5 gene or protein in the preparation of products for detecting and diagnosing whether pigs are infected with PRRSV.

[0017] In actual work, the diagnostic criterion is: an abnormally increased expression level of MARCH5 indicates infection with PRRSV or suffering from PRRS. The "abnormally increased" mentioned here is based on the general level of MARCH5 expression in pigs in the art.

[0018] The application of a detection reagent for MARCH5 gene or protein in screening PRRS-resistant pigs or in the preparation of products for differentiating the susceptibility of pigs to PRRSV.

[0019] The application of a detection reagent for MARCH5 gene or protein in the preparation of products for screening therapeutic drugs against PRRS in pigs.

[0020] In actual work, the screening criterion is: the higher the expression level of MARCH5 in pigs, the stronger the resistance to PRRSV and the less susceptible; conversely, the more susceptible.

[0021] In addition, as a preferable implementation scheme, the present invention provides a primer for detecting the expression of MARCH5 in pigs, and the sequence is shown in SEQ ID NO.1-2. Specifically, the upstream primer - F (SEQ ID NO.1): 5′-GTGACAGTGATGCAGGTTGT-3′; the downstream primer MARCH5 - R (SEQ ID NO.2): 5′-ATTTGCGCCACAGTCTAAGC-3′.

[0022] Specifically, the present invention also provides a method for screening pigs resistant to porcine reproductive and respiratory syndrome (PRRS), which discriminates the resistance or susceptibility of pigs to PRRS virus by detecting the expression level (expression quantity) of MARCH5 in pigs. Further, the discrimination criterion is: pigs with a high expression quantity of MARCH5 are resistant to PRRS virus; conversely, they are susceptible.

[0023] In specific operations, the high or low expression quantity of MARCH5 is based on the general level of MARCH5 expression quantity in pigs in the art. The high or low here is relative. According to whether the expression quantity of MARCH5 is abnormally high or low, it is determined whether a pig may be resistant to PRRSV and the degree of susceptibility to PRRSV.

[0024] More specifically, based on the results of the experiments of the present invention, the specific data criterion for discrimination is approximately: when the expression quantity of MARCH5 is higher than 4.5 ± 0.5 × 10 -2 (relative to the internal reference HPRT1), the pig is resistant to the virus; when the expression quantity is lower than 4.5 ± 0.5 × 10 -2 (relative to the internal reference HPRT1), it is susceptible.

[0025] Particularly, the present invention does not strictly limit the detection method for the expression quantity of MARCH5, and it can be an existing gene expression quantity detection method in the art.

[0026] In addition, the present invention also provides:

[0027] The application of an expression promoter or expression activator of MARCH5 gene or protein in the preparation of a preparation for improving the resistance of pigs to PRRS virus.

[0028] The application of an expression promoter or expression activator of MARCH5 gene or protein in the preparation of a therapeutic drug for porcine reproductive and respiratory syndrome or in the preparation of an antiviral drug against PRRS virus.

[0029] The present invention also provides a preparation for improving the resistance of pigs to PRRS virus, which contains an expression promoter or expression activator of MARCH5 gene or protein.

[0030] The "expression promoter or expression activator" described herein includes, but is not limited to, enzyme activators, compound promoters, plasmids, etc.

[0031] In addition, the present invention also provides the application of MARCH5 gene in the cultivation of pig breeds resistant to PRRS.

[0032] Specifically, a method for cultivating pigs resistant to PRRS is to overexpress the MARCH5 gene of pigs.

[0033] As an alternative implementation, the method for cultivating pigs resistant to PRRS can adopt gene editing technology.

[0034] The present invention has the following beneficial effects:

[0035] The present invention discloses the application of MARCH5 in identifying the susceptibility of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) and in preparing drugs. The research of the present invention shows that the expression level of MARCH5 in pigs is closely related to PRRSV infection. After PRRSV infects porcine alveolar macrophages (PAMs) cells, the mRNA and protein levels of MARCH5 increase significantly. Interference with MARCH5 promotes PRRSV replication, while overexpression of MARCH5 inhibits virus replication. When the expression level of MARCH5 in pigs is relatively high, pigs are resistant to PRRSV; conversely, they are susceptible. Therefore, MARCH5 can be used as a criterion for determining the susceptibility or resistance to PRRSV. Pigs with abnormally high expression levels of MARCH5 are resistant to PRRSV, while those with low expression levels are susceptible. The present invention lays a solid foundation for identifying the susceptibility of pigs to PRRSV and for the screening, identification, and breeding of PRRSV-resistant pigs, which is beneficial to the cultivation of excellent pig strains.

[0036] The present invention lays a foundation for screening / identifying / selectively breeding PRRSV-resistant pigs and for PRRSV disease-resistant breeding. It has good clinical application value for the screening and cultivation of PRRSV-resistant pigs, is of great significance for the prevention and control of porcine reproductive and respiratory syndrome, and is also beneficial to the cultivation of excellent pig strains. At the same time, the present invention also provides a new application for MARCH5. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It shows the protein expression level of MARCH5 at different time points after PRRSV infects PAMs cells.

[0038] Figure 2 It shows the mRNA expression level of MARCH5 at different time points after PRRSV infects PAMs cells.

[0039] Figure 3 It shows the protein expression levels of MARCH5 and PRRSV-N at different time points after virus infection following siRNA interference of MARCH5.

[0040] Figure 4 It shows the determination of virus titers at different time points after virus infection following siRNA interference of MARCH5 (in the figure, Si-Ctrl is the siRNA negative control, and Si-694 is the siRNA targeting MARCH5).

[0041] Figure 5 It shows the protein expression levels of MARCH5 and PRRSV-N at different time points after virus infection following overexpression of MARCH5.

[0042] Figure 6 It shows the determination of virus titers at different time points after virus infection following overexpression of MARCH5. Detailed implementation mode

[0043] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments. However, the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0045] Statistical analysis of the following examples of the present invention: All experiments were independently repeated at least 3 times, and the results were expressed as the mean and standard error. One-way ANOVA and T-tests were used for analysis. All statistical analyses were performed using P < 0.05 as the test criterion for significant statistical differences. The analysis software was SPSS 16.0 and GraphPad Prism 5.

[0046] Example 1 Expression of MARCH5 gene

[0047] Porcine alveolar macrophages (PAMs) were cultured in 1640 medium containing 10% fetal bovine serum, and inoculated with the highly pathogenic PRRSV strain Li11 (this strain is stored in our laboratory) at an MOI of 0.1. The cells were then continuously cultured in 1640 medium containing 2% fetal bovine serum at 37°C. Cells were collected at different time points of culture: 0, 6, 12, 24, 36 h (where 0 h means no virus infection). The primers for the MARCH5 gene were used: forward primer - F (the sequence is shown in SEQ ID NO.1): 5′-GTGACAGTGATGCAGGTTGT-3′; reverse primer MARCH5 - R (the sequence is shown in SEQ ID NO.2): 5′-ATTTGCGCCACAGTCTAAGC-3′, and real-time PCR and Western-blot were performed for detection.

[0048] The results of the expression of MARCH5 protein and mRNA after PRRSV infection of PAMs cells are as Figure 1 and Figure 2 shown, indicating that after PRRSV infection of PAMs cells, the expression levels of MARCH5 protein and mRNA increased significantly. As the infection time prolonged, the expression level of PRRSV-N protein increased more significantly.

[0049] Example 2 Interference of MARCH5

[0050] Design MARCH5 siRNA primers (MARCH5-694 sense: GCAGGUUGUAGGCCAUAAATT; antisense: UUUAUGGCCUACAACCUGCTT) and a pair of Negative Control (Si-Ctrl sense: GGCCCCUUAUGUTTAGCCUAA; antisense: GTTAGUUGUCGGAUCCAGACA) (siRNA negative control) primers, and send them to Thermo Fisher Scientific for synthesis. Then use Lipofectamine TM RNAiMAX transfection reagent (purchased from Thermo Fisher Scientific) to culture PAMs cells in 1640 medium containing 10% fetal bovine serum in two 6-well plates until 80% confluence. Take one 6-well plate, and dilute siRNA-694, Si-Ctrl and Lipofectamine TM RNAiMAX with Opti-MEM Reduced Serum Medium; mix them in a 1:1 ratio after dilution and incubate at room temperature for 5 min, then add them to PAMs cells replaced with fresh 1640 medium and culture in a 37°C, 5% CO2 incubator for 6 h. Discard the supernatant, wash once with PBS, and continue to culture with 1640 medium containing 10% fetal bovine serum for 48 h, then collect the cells. Then verify the interference effect of MARCH5 by qRT-PCR.

[0051] After culturing in another 6-well plate for 48 h, add PRRSV to the medium, and then culture for 12, 24, 36 h. Collect the cells and verify the effect of MARCH5 interference on PRRSV replication by Western-blot and TCID50 assays.

[0052] The detection results are as Figure 3 shown. After MARCH5 was interfered with siRNA, compared with the control group, siRNA significantly inhibited the expression of MARCH5 protein. After PRRSV infection for 12, 24, 36 h, the expression level of PRRSV-N protein increased significantly.

[0053] The TCID50 detection results are as Figure 4 shown, showing that after MARCH5 interference, the PRRSV virus titer increased significantly. It indicates that after MARCH5 was interfered with siRNA, it promoted the replication of PRRSV.

[0054] Example 3 Overexpression of MARCH5

[0055] The pcDNA3.1-MARCH5 overexpression vector was constructed using restriction enzyme ligation technology. PAMs cells were cultured in 1640 medium containing 10% fetal bovine serum in two 6-well plates until 80% confluence. In one 6-well plate, Lipofectamine TM 2000 (purchased from Thermo Fisher Scientific) was used to transfer pcDNA3.1-MARCH5 and pcDNA3.1 empty vector into PAMs cells respectively. The cells were cultured in an incubator at 37°C and 5% CO2 for 6 h, then the supernatant was discarded, and the cells were washed once with PBS and continued to be cultured in 1640 medium containing 10% fetal bovine serum for 24 h. The cells were collected to detect the overexpression effect.

[0056] After culturing for 24 h in another 6-well plate, PRRSV was added to the medium, and then the cells were cultured for another 12, 24, 36 h. The cells were collected to detect the effect of MARCH5 overexpression on PRRSV replication.

[0057] The detection results are as Figure 5 shown. After MARCH5 overexpression, the protein level of MARCH5 increased significantly. After PRRSV infection for 12, 24, 36 h, the expression level of PRRSV-N protein decreased significantly.

[0058] The results of TCID50 determination are as Figure 6 shown, indicating that the virus titer of PRRSV decreased significantly. It shows that MARCH5 overexpression inhibits PRRSV replication.

[0059] Example 4 Application of MARCH5 in identifying the susceptibility of pigs to porcine reproductive and respiratory syndrome virus

[0060] Local Chinese pig breeds Tongcheng pigs and Meishan pigs, and foreign pig breeds Landrace pigs and Yorkshire pigs were used as research objects. Among them, it is known that Tongcheng pigs and Meishan pigs are not susceptible to porcine reproductive and respiratory syndrome virus, that is, they have resistance; while Landrace pigs and Yorkshire pigs are more susceptible to porcine reproductive and respiratory syndrome virus.

[0061] Ten Tongcheng pigs, ten Meishan pigs, ten Landrace pigs, and ten Yorkshire pigs were selected. Five pigs were in one group, that is, divided into two groups. One group was challenged with PRRSV, and the other group was not treated (control). Blood samples were collected 1 day before challenge and on the 7th, 14th, 21st, 28th days after challenge. The control group collected blood samples synchronously. The pigs were sacrificed on the 28th day, and blood, lung, and lymph node tissues were collected. A part of the serum was used for TCID50 determination, and the remaining serum, as well as lung and lymph node tissues, were used to extract RNA, reverse transcribe, and perform qRT-PCR detection on MARCH5 and PRRSV N protein, and pathological sections of lung tissues were made.

[0062] Determine the susceptibility of local breeds of Tongcheng pigs and Meishan pigs and foreign breeds of Landrace pigs and Yorkshire pigs to PRRSV according to the TCID50, PRRSV N protein expression level, and lung tissue pathological sections. Study the relationship between the expression level of MARCH5 in pigs and the disease resistance of the above four breeds of pigs to PRRSV.

[0063] According to the results, it can be judged that Tongcheng pigs and Meishan pigs are not susceptible (resistant) to porcine reproductive and respiratory syndrome virus (PRRSV), while Landrace pigs and Yorkshire pigs are more susceptible to PRRSV.

[0064] At the same time, the detection results of the expression level of MARCH5 in pigs before virus challenge (gene background expression level) showed that the expression level of MARCH5 in Tongcheng pigs and Meishan pigs was significantly higher than that in Landrace pigs and Yorkshire pigs; after virus challenge, the expression level of MARCH5 in pigs increased significantly, which was consistent with the experimental results at the cellular level.

[0065] In addition, based on a large number of experimental results, the specific data standard for identifying the susceptibility of pigs to PRRSV is roughly as follows: when the expression level of MARCH5 is higher than 4.5 ± 0.5 × 10 -2 (relative to the internal reference HPRT1 gene), pigs are resistant to the virus; when the expression level is lower than 4.5 ± 0.5 × 10 -2 (relative to the internal reference HPRT1 gene), they are susceptible. And in pigs, compared with other pathogen infections, after pigs are infected with PRRSV, the expression level of MARCH5 increases significantly, suggesting that MARCH5 is closely related to PRRSV infection.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. The use of a reagent for detecting the expression level of the MARCH5 gene or protein in the preparation of a product for detecting and diagnosing porcine reproductive and respiratory syndrome.

2. The use of a reagent for detecting the expression level of the MARCH5 gene or protein in the preparation of a product for detecting and diagnosing whether a pig is infected with porcine reproductive and respiratory syndrome virus.

3. Use of a reagent for detecting the expression level of the MARCH5 gene or protein in the preparation of a product for differentiating the susceptibility of pigs to porcine reproductive and respiratory syndrome virus, characterized in that, The specific identification method is as follows: perform qRT-PCR detection relative to the internal reference HPRT1 gene. When the expression level of MARCH5 is higher than 4.5 ± 0.5 × 10 -2 , it is a resistant pig; when the expression level is lower than 4.5 ± 0.5 × 10 -2 , it is a susceptible pig.

4. Use of an expression promoter or expression activator of MARCH5 gene or protein in the preparation of a preventive drug for porcine reproductive and respiratory syndrome, characterized in that, The expression promoter or expression activator is an overexpression vector of MARCH5.

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