Application of IL-8 protein in porcine reproductive and respiratory syndrome virus infection

Abstract

The invention discloses application of IL-8 protein in porcine reproductive and respiratory syndrome virus infection, and belongs to the technical field of veterinary biological products. Researches show that IL-8 which indirectly influences the PRRSV infection course by regulating inflammatory response in the prior art also has the effect of directly inhibiting PRRSV. The IL-8 protein can significantly reduce highly pathogenic HP-PRRSV, low pathogenic LP-PRRSV, NADC30-like and recombinant PRRSV infections, and can be developed into drugs for preventing and treating different types of PRRSV infections, so that a brand new thought is provided for PRRS prevention and treatment, the problem that the efficacy of therapeutic drugs is reduced or invalid due to high PRRSV variability is effectively solved, and the application prospect is broad. And the method is of great significance to actual production.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary biological products, and in particular to the application of IL-8 protein in porcine reproductive and respiratory syndrome virus infection. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV). The main clinical features of the disease are reproductive disorders in sows and breathing difficulties in pigs of all ages, causing huge economic losses to the pig industry. Due to the high variability of PRRSV, multiple types of PRRSV are prevalent in clinical practice, with HP-PRRSV, NADC30-like and LP-PRRSV being the dominant strains. The existing vaccine immunity is not ideal, and there is no effective treatment drug, which has become a difficult problem in the prevention and control of the disease.

[0003] The protein encoded by the CXCL8 gene is a member of the CXC chemokine family and is a major mediator of inflammatory responses. The encoded protein is often referred to as interleukin-8 (IL-8). IL-8 is secreted by mononuclear macrophages, neutrophils, eosinophils, T lymphocytes, epithelial cells, and fibroblasts. It acts as a chemokine by guiding neutrophils to the site of infection. It is worth noting that IL-8, as a common inflammatory factor, can participate in the pro-inflammatory signaling cascade with other inflammatory cytokines and play a role in systemic inflammatory response syndrome (SIRS). Existing technologies have focused on regulating the immune response, and any viral infection will affect its expression.

[0004] For porcine reproductive and respiratory syndrome virus infection, current studies have shown that the effect of IL-8 on the course of PRRSV infection is indirectly affected by regulating the immune response, and it mainly plays a pro-inflammatory role in PRRSV infection. Specifically, PRRSV virus promotes IL-8 gene transcription by activating signaling pathways such as NF-κB, significantly upregulating IL-8 secretion. PRRSV mainly targets macrophages, while the target of IL-8 (neutrophils) is not the main host cell of the virus. Therefore, it is unclear whether IL-8 can directly inhibit the effect of PRRSV, and there are currently no reports on the use of IL-8 protein as a drug for the prevention and treatment of PRRSV infection. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of IL-8 protein in porcine reproductive and respiratory syndrome virus infection. In the present invention, from the perspective of direct binding of IL-8 protein to viral protein, it is found that IL-8 can neutralize PRRSV and has a significant inhibitory effect on its infection. At the same time, it has a significant blocking effect on different types of PRRSV infection. Therefore, IL-8 can be used as an effective drug for PRRS.

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

[0007] In a first aspect, the present invention provides the use of IL-8 protein in the preparation of a drug for preventing and treating porcine reproductive and respiratory syndrome virus infection.

[0008] The IL-8 protein directly inhibits porcine reproductive and respiratory syndrome virus infection by neutralizing porcine reproductive and respiratory syndrome virus.

[0009] The porcine reproductive and respiratory syndrome virus includes highly pathogenic HP-PRRSV, low pathogenic LP-PRRSV, NADC30-like and recombinant PRRSV.

[0010] The recombinant PRRSV is formed by the recombination of NADC30-like and LP-PRRSV.

[0011] The medicine for preventing and treating porcine reproductive and respiratory syndrome virus infection takes IL-8 protein as an effective component.

[0012] The IL-8 protein is a monkey-derived IL-8 protein or a porcine-derived IL-8 protein, and the amino acid sequence of the monkey-derived IL-8 protein is shown in SEQ ID NO.1, which is as follows:

[0013] SEQ ID NO. 1: MTSKLAVALLAAFLLSAALCEGAVLPRSAKELRCQCIKTYSKPIHP KFIKELRVIESGPHCVNTEIIVKLSDGRELCLDPKVPWVSRVVEKFLKRAESQNS.

[0014] The amino acid sequence of the porcine IL-8 protein is shown in SEQ ID NO.2, which is as follows:

[0015] SEQ ID NO. 2: MTSKLAVAFLAVFLLSAALCEAAVLARVSAELRCQCINTHSTPFHP KFIKELRVIESGPHCENSEIIVKLVNGKEVCLDPKEKWVQKVVQIFLKRTEKQQQQQ.

[0016] The IL-8 protein can be induced by a prokaryotic expression system or a eukaryotic expression system and obtained by protein purification, or can be directly obtained by chemical synthesis.

[0017] Beneficial effects of the present invention:

[0018] The present invention study shows that IL-8, which indirectly affects the course of PRRSV infection by regulating inflammatory response in the prior art, also has the effect of directly inhibiting PRRSV. This study proves that IL-8 protein can significantly reduce the infection of highly pathogenic HP-PRRSV, low pathogenic LP-PRRSV, NADC30-like and recombinant PRRSV, and can be developed into drugs for preventing and treating different types of PRRSV infection, thus providing a new idea for PRRS prevention and treatment, effectively solving the problem of reduced efficacy or ineffectiveness of therapeutic drugs caused by the high variability of PRRSV, which is of great significance for actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the interaction between IL-8 protein and PRRSV structural protein; wherein, Figure 1 A in the middle is Western blot verification of interacting proteins. Figure 1 In B, the direct interaction of proteins was verified by Western blot.

[0020] Figure 2 IL-8 protein neutralizes PRRSV.

[0021] Figure 3 The effect of IL-8 protein on TA-12 infection on Marc-145; Figure 3 Middle A is the detection of cytotoxicity of IL-8 protein to Marc-145 cells; Figure 3 Middle B is the real-time PCR detection of IL-8 protein after TA-12 infection on Marc-145; Figure 3 Middle C is the result of the IL-8 protein titer test on Marc-145 after TA-12 infection; Figure 3 D in the middle is the Western blot detection result of IL-8 protein after TA-12 infection on Marc-145, and N is the N protein of PRRSV.

[0022] Figure 4 The effect of IL-8 protein on TA-01, TA-02 and CH-1R infection on Marc-145; Figure 4 Center A shows the Western blot detection results of IL-8 protein after infection of TA-01, TA-02, and CH-1R on Marc-145; Figure 4 Middle B, Figure 4 Middle C, Figure 4 D in the middle are the real-time PCR detection results of IL-8 protein on Marc-145 after infection with TA-01, TA-02 and CH-1R; Figure 4 Middle E, Figure 4 Medium F, Figure 4 G in the middle are the results of virus titer detection of IL-8 protein after infection of TA-01, TA-02 and CH-1R on Marc-145.

[0023] Figure 5 :The effect of IL-8 protein on TA-12 infection on PAM; Among them, Figure 5 Middle A is the detection of IL-8 protein cytotoxicity to PAM cells; Figure 5 Middle B is the real-time PCR detection of IL-8 protein on PAM after TA-12 infection; Figure 5 Middle C is the result of the virus titer test of IL-8 protein on PAM after TA-12 infection; Figure 5 D in the middle is the Western blot detection result of IL-8 protein after TA-12 infection on Marc-145, and N is the N protein of PRRSV.

[0024] Figure 6 :The effect of IL-8 protein on PAM on TA-01, TA-02 and CH-1R infection; Among them, Figure 6 Center A is the Western blot detection result of IL-8 protein on PAM after infection with TA-01, TA-02, and CH-1R; Figure 6 Middle B, Figure 6 Middle C, Figure 6 Middle D shows the real-time PCR detection results of IL-8 protein on PAM after infection with TA-01, TA-02 and CH-1R; Figure 6 Middle E, Figure 6 Medium F, Figure 6 Middle G shows the virus titer detection results of IL-8 protein on PAM after infection of TA-01, TA-02 and CH-1R. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0026] As mentioned above, PRRSV infection is often accompanied by immunosuppression (such as blocked IFN secretion) and inflammatory storm (such as increased IL-8). The current existing technology believes that IL-8 mainly participates in the immune response as a pro-inflammatory factor and indirectly affects the process of PRRSV virus infection, rather than being a direct antiviral molecule.

[0027] The present invention experimentally verifies that IL-8 can also directly inhibit PRRSV infection, overturning the traditional understanding of the function of chemokines. This study clarifies the dual role of IL-8, lays a foundation for promoting in-depth research on the antiviral mechanisms of IL-8 and other chemokines, provides a supplementary means for the direct use of IL-8 or overexpression of IL-8 at the infection site through a gene delivery system to treat PRRS, and provides a key target for the development of new antiviral drugs.

[0028] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0029] The experimental materials used in the examples of the present invention without specific description are all conventional experimental materials in the field and can be purchased through commercial channels. Among them:

[0030] The PRRSV strains used in the present invention are represented by TA-12 strains for HP-PRRSV type, CH-1R strains for LP-PRRSV type, TA-01 strains for NADC30-like type, and TA-02 strains for PRRSV recombinant virus (recombination of NADC30-Like and HP-PRRSV). The strains used above are all existing strains.

[0031] The maintenance medium used in the present invention is a culture medium containing 2% fetal bovine serum.

[0032] If no specific experimental conditions and methods are specified in the examples of the present invention, conventional conditions are generally used, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0033] Example 1: Interaction between IL-8 protein and PRRSV structural protein

[0034] IL-8 and the main surface proteins of PRRSV (GP2, GP3, GP4, GP5 and M) and its empty vector GFP-C1 were co-transfected on 293T cells. Cell samples were collected after 24 hours, and the supernatant was collected as a total protein extract after lysis. Anti-GFP tag monoclonal antibody was added to the total protein extract and incubated for 2 hours. Protein A / G magnetic beads were added and incubated overnight. After washing and elution, the interacting proteins were verified by Western blot ( Figure 1 In A). To further verify that there is a direct interaction between IL-8 and viral proteins, the main surface proteins of PRRSV (GP2, GP3, GP4, GP5 and M) and its empty vector GFP-C1 were transfected into 293T cells. Cell samples were collected after 24 hours, and the supernatant was collected as a total protein extract after lysis. Then the His-tagged porcine IL-8 protein was used as a bait protein and incubated with Ni-NTA resin at 4°C for 3 hours, and then the total protein extract was mixed with it and incubated at 4°C for 4 hours. After washing with washing solution several times, the eluent was eluted and verified by Western blot ( Figure 1 Middle B), the results show that IL-8 can directly bind to the structural proteins of PRRSV virus.

[0035] Furthermore, the monkey IL-8 protein was diluted to 60 μg / mL with 2% maintenance solution, and then TA-12 was added to the mixture at an MOI of 0.01, incubated at 37°C for 1 hour, and inoculated onto a monolayer of Marc-145 cells. After 24 hours, the cells were collected to extract RNA, and reverse transcribed for fluorescence quantitative PCR verification (primers used for fluorescence quantitative PCR are shown in Table 1) to detect the number of viral copies to determine the neutralization effect of IL-8 protein on PRRSV. Real-time PCR detection results showed that 60 μg / ml IL-8 protein had a neutralizing effect on HP-PRRSV ( Figure 2 ), the experimental results showed that IL-8 protein can block virus adsorption and thus play a virus neutralizing role.

[0036] Example 3: Blocking experiment of IL-8 protein on PRRSV infection in Marc-145

[0037] In order to verify whether IL-8 protein can regulate PRRSV infection as a direct antiviral molecule, the toxicity of different concentrations of monkey IL-8 protein (0.1μg / ml, 0.5μg / ml, 1μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, 100μg / ml) on Marc-145 cells was verified by CCK-8 method. The results showed that IL-8 protein was within the safe application range between 0-80μg / ml ( Figure 3 (A).

[0038] 1. Blocking experiment of TA-12 infection

[0039] The monkey IL-8 protein was prepared into a series of concentrations (0, 30, 60, 90 μg / mL) with maintenance solution, of which the 0 μg / mL group used pure maintenance solution as a blank control, and then TA-12 was added to the mixture at an MOI of 0.01, and inoculated on a monolayer of Marc-145 cells. After 24 hours of TA-12 infection, the cell supernatant was collected, and the cells were washed with pre-cooled PBS, and all the cells were blown down with a pipette, transferred to a new centrifuge tube, centrifuged at 2000 rpm for 3 minutes, and the supernatant was discarded, leaving the precipitate. The cells were lysed with cell lysis solution to collect the protein for Western blot verification, and the virus titer was determined with the cell supernatant. At the same time, the cell RNA was extracted and reverse transcribed for fluorescence quantitative PCR verification (the primers used for fluorescence quantitative PCR are shown in Table 1) to detect the effect of different concentrations of IL-8 protein on TA-12 infection.

[0040] Real-time PCR results showed that 60 μg / ml IL-8 protein could reduce the copy number of HP-PRRSV in Marc-145 cells by 5 times ( Figure 3 (B) Virus titer determination results also showed that 60 μg / ml IL-8 protein could increase the TCID of HP-PRRSV 50 Reduce 1 lg value ( Figure 3 Western blot results also showed that 60 μg / ml IL-8 protein could significantly inhibit the infection of HP-PRRSV in Marc-145 cells ( Figure 3 Middle D), the above results show that IL-8 protein can block the infection of HP-PRRSV on Marc-145 cells.

[0041] Table 1: Primers used for fluorescence quantification

[0042]

[0043] Note: In Table 1, Marc-145 cell line is a PRRSV susceptible cell line derived from monkey kidney epithelial cells, and PAM is a primary porcine lung macrophage. The primer sequences used to detect GAPDH in these two cell lines are different.

[0044] 2. Blocking experiment of TA-01, TA-02 and CH-1R infection

[0045] The monkey IL-8 protein was prepared into a series of concentrations (0, 30, 60, 90 μg / mL) with maintenance solution, of which the 0 μg / mL group used pure maintenance solution as a blank control, and then TA-01, TA-02 and CH-1R were added and mixed at an MOI of 0.01 and inoculated on a monolayer of Marc-145 cells. The cell supernatant was collected 24 hours after virus infection, and the cells were washed with pre-cooled PBS. All cells were blown down with a pipette and transferred to a new centrifuge tube, centrifuged at 2000 rpm for 3 minutes, and the supernatant was discarded, leaving the precipitate. The cells were lysed with cell lysis solution to collect the protein for Western blot verification, and the cell RNA was extracted and reverse transcribed for fluorescence quantitative PCR verification (the primers used for fluorescence quantitative PCR are shown in Table 1), and the cell supernatant was used to determine the virus titer to detect the effects of different concentrations of IL-8 protein on TA-01, TA-02 and CH-1R infection.

[0046] Western blot results showed that 60 μg / ml IL-8 protein could significantly inhibit the infection of three different strains in Marc-145 cells ( Figure 4 A), Real-time PCR results showed that 60 μg / ml IL-8 protein could reduce the copy number of three different strains in Marc-145 cells ( Figure 4 Middle B, Figure 4 Middle C and Figure 4 (D) Virus titer determination results also showed that 60 μg / ml IL-8 protein could increase the TCID of three different strains. 50 At least 1 lg value is reduced ( Figure 4 Middle E, Figure 4 Medium F and Figure 4 The above results indicate that IL-8 protein can block the infection of LP-PRRSV, NADC30-like and PRRSV recombinant strains on Marc-145 cells.

[0047] Example 4: Blocking experiment of IL-8 protein on PAM against PRRSV infection

[0048] The toxicity of different concentrations of porcine IL-8 protein (0.1μg / ml, 0.5μg / ml, 1μg / ml, 5μg / ml, 10μg / ml, 20μg / ml, 40μg / ml, 60μg / ml, 80μg / ml, 100μg / ml) to PAM cells was verified by CCK-8 method. The results showed that IL-8 protein was within the safe application range between 0-40μg / ml ( Figure 4 (A).

[0049] 1. Blocking experiment of TA-12 infection

[0050] The porcine IL-8 protein was prepared into a series of concentrations (0, 2, 5, 10, 20, 40 μg / mL) with maintenance solution, of which the 0 μg / mL group used pure maintenance solution as a blank control, and then TA-12 was added to the mixture at an MOI of 0.01 and inoculated onto a monolayer of PAM cells. After 24 hours of TA-12 infection, the cell supernatant was collected, all cells were blown down with a pipette, transferred to a new centrifuge tube, centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and the precipitate was retained. The cells were lysed with cell lysis solution to collect the protein for Western blot verification, and the virus titer was determined with the cell supernatant. At the same time, the cell RNA was extracted and reverse transcribed for fluorescence quantitative PCR verification (the primers used for fluorescence quantitative PCR are shown in Table 1) to detect the effect of different concentrations of IL-8 protein on TA-12 infection.

[0051] Real-time PCR results showed that 40 μg / ml IL-8 protein could reduce the copy number of HP-PRRSV in Marc-145 cells by 10 times ( Figure 5 (B) Virus titer determination results also showed that 40 μg / ml IL-8 protein could increase the TCID of HP-PRRSV 50 Reduce 2 lg value ( Figure 5 Western blot results also showed that 40 μg / ml IL-8 protein could significantly inhibit the infection of HP-PRRSV in PAM cells ( Figure 5 (D) The above results indicate that IL-8 protein can block the infection of HP-PRRSV on PAM cells.

[0052] 2. Blocking experiment of TA-01, TA-02 and CH-1R infection

[0053] The porcine IL-8 protein was prepared into a series of concentrations (0, 2, 5, 10, 20, 40 μg / mL) with maintenance solution, of which the 0 μg / mL group used pure maintenance solution as a blank control, and then TA-01, TA-02 and CH-1R were added and mixed at an MOI of 0.01 and inoculated onto a monolayer of PAM cells. After 24 hours of viral infection, the cell supernatant was collected, all cells were blown down with a pipette, transferred to a new centrifuge tube, centrifuged at 2000 rpm for 3 minutes, the supernatant was discarded, and the precipitate was retained. The cells were lysed with cell lysis solution to collect the protein for Western blot verification, and the cell RNA was extracted and reverse transcribed for fluorescence quantitative PCR verification (the primers used for fluorescence quantitative PCR are shown in Table 1), and the cell supernatant was used to determine the virus titer to detect the effects of different concentrations of IL-8 protein on TA-01, TA-02 and CH-1R infection.

[0054] Western blot results also showed that 40 μg / ml IL-8 protein could significantly inhibit the infection of three different strains in PAM cells ( Figure 6 A), Real-time PCR results showed that 40 μg / ml IL-8 protein could reduce the copy number of three different strains on PAM cells ( Figure 6 Middle B, Figure 6 Middle C, Figure 6 (D) Virus titer determination results also showed that 40 μg / ml IL-8 protein could increase the TCID of three different strains. 50 At least 1 lg value is reduced ( Figure 6 Middle E, Figure 6 Medium F, Figure 6 The above results indicate that IL-8 protein can block the infection of LP-PRRSV, NADC30-like and PRRSV recombinant strains on PAM cells.

[0055] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of IL-8 protein in the preparation of drugs for preventing and treating porcine reproductive and respiratory syndrome virus infection.

2. The use according to claim 1, characterized in that: The IL-8 protein directly inhibits porcine reproductive and respiratory syndrome virus infection by neutralizing porcine reproductive and respiratory syndrome virus.

3. The use according to claim 1, characterized in that: The porcine reproductive and respiratory syndrome virus includes highly pathogenic HP-PRRSV, low pathogenic LP-PRRSV, NADC30-like and recombinant PRRSV.

4. The use according to claim 3, characterized in that: The recombinant PRRSV is formed by the recombination of NADC30-like and LP-PRRSV.

5. The use according to claim 1, characterized in that: The medicine for preventing and treating porcine reproductive and respiratory syndrome virus infection takes IL-8 protein as an effective ingredient.

6. The use according to any one of claims 1 to 5, characterized in that: The IL-8 protein is a monkey IL-8 protein or a pig IL-8 protein. The amino acid sequence of the monkey IL-8 protein is shown in SEQ ID NO.1, and the amino acid sequence of the pig IL-8 protein is shown in SEQ ID NO.2.

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