Application of RPL34 protein in porcine delta coronavirus

Abstract

The present application relates to the field of biotechnology, and in particular to an application of RPL34 protein in porcine delta coronavirus; the application includes: using overexpressed RPL34 protein as a biomarker for porcine delta coronavirus infection in an organism; through a large number of experiments, it was found that overexpression of RPL34 protein has a promoting effect on the infection process of porcine delta coronavirus, and in addition, the expression level of RPL34 protein was found to be significantly increased in cells infected with porcine delta coronavirus, which indicates that the expression of RPL34 protein can be used as a type of biomarker for porcine delta coronavirus infection in the organism; in addition, the use of the inhibitor drug XL019 and siRNA‑RPL34 that can knock down RPL34 to inhibit the expression of RPL34 can inhibit the infection level of porcine delta coronavirus, thereby filling the gap between RPL34 protein and porcine delta coronavirus infection.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an application of RPL34 protein in porcine deltacoronavirus. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is an enteropathogenic coronavirus that causes watery diarrhea in pigs. It is infectious to poultry and even humans, posing a significant threat to the swine industry and public health. Therefore, understanding the infection and pathogenicity mechanisms of PDCoV and developing targeted treatments are urgent tasks for its prevention and control.

[0003] RPL34 is a ribosomal protein composed of 110 amino acids and is also a component protein of the ribosome. It is involved in the self-assembly of the ribosome and the synthesis of proteins. However, there is currently a lack of research on the association between RPL34 protein and porcine delta coronavirus infection. Summary of the Invention

[0004] The present application provides an application of RPL34 protein in porcine deltacoronavirus to solve the following technical problem: how to fill the gap between the association between RPL34 protein and porcine deltacoronavirus infection.

[0005] In a first aspect, the present application provides an application of RPL34 protein as a biomarker of porcine deltacoronavirus, wherein the application includes: using overexpressed RPL34 protein as a biomarker of porcine deltacoronavirus infection in the body.

[0006] Optionally, the expression level of the overexpressed RPL34 protein is positively correlated with the degree of infection of the porcine delta coronavirus.

[0007] Optionally, the expression level of the overexpressed RPL34 protein is 5 times or more higher than the expression level of the normal RPL34 protein.

[0008] In a second aspect, the present application provides a therapeutic drug for porcine deltacoronavirus, wherein the therapeutic drug includes an inhibitor that inhibits the RPL34 protein.

[0009] Optionally, the inhibitor comprises the inhibitor drug XL019.

[0010] Optionally, the concentration of the inhibitor drug XL019 is ≥5 μM.

[0011] Optionally, the inhibitor comprises siRNA-RPL34.

[0012] Optionally, the concentration of the siRNA-RPL34 is ≥10 μg / well.

[0013] Optionally, the preparation method of the siRNA-RPL34 comprises:

[0014] PCR amplification of the target gene fragment with RPL34-related gene is performed, and then recovered to obtain the amplified product;

[0015] Cloning the amplified product into an expression vector to obtain a recombinant plasmid;

[0016] Prompting the recombinant plasmid to express siRNA-RPL34;

[0017] Wherein, the amplification primer set used for the PCR amplification includes a first primer set, a second primer set, a third primer set and a transcription primer set;

[0018] The forward primer of the first primer set is shown as SEQ ID NO.1, and the reverse primer of the first primer set is shown as SEQ ID NO.2;

[0019] The forward primer of the second primer set is shown as SEQ ID NO.3, and the reverse primer of the second primer set is shown as SEQ ID NO.4;

[0020] The forward primer of the third primer set is shown as SEQ ID NO.5, and the reverse primer of the third primer set is shown as SEQ ID NO.6;

[0021] The forward primer of the transcription primer set is shown as SEQ ID NO.7, and the reverse primer of the transcription primer set is shown as SEQ ID NO.8.

[0022] In a third aspect, the present application provides a method for inhibiting porcine delta coronavirus infection for non-diagnostic and therapeutic purposes, the method comprising:

[0023] Knock out genes related to the expression of RPL34 protein in the body; and / or

[0024] Use of inhibitors that inhibit RPL34 protein expression;

[0025] Wherein, when the method includes using an inhibitor to inhibit the expression of RPL34 protein, the inhibitor includes the inhibitor drug XL019 and / or siRNA-RPL34.

[0026] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0027] The present application embodiment provides an application of RPL34 protein as a biomarker of porcine delta coronavirus, and the application includes: using overexpressed RPL34 protein as a biomarker of porcine delta coronavirus infection. Through a large number of experiments, it was found that overexpression of RPL34 protein has a promoting effect on the infection process of porcine delta coronavirus. In addition, the expression level of RPL34 protein was found to be significantly increased in cells infected with porcine delta coronavirus, which indicates that the expression of RPL34 protein can be used as a type of biomarker for porcine delta coronavirus infection of the body. In addition, the use of inhibitor drug XL019 and siRNA-RPL34 that can knock down RPL34 to inhibit the expression of RPL34 can inhibit the infection level of porcine delta coronavirus, thereby filling the gap between RPL34 protein and porcine delta coronavirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0029] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0030] Figure 1 This is a graph showing the electrophoresis results of the PCR amplification products of the RPL34 gene provided in the examples of the present application, wherein: Figure 1 A is the electrophoresis result of the PCR amplification product of RPL34-H. Figure 1 B is the electrophoresis result of the PCR amplification product of RPL34-M. Figure 1 C is the electrophoresis result of the PCR amplification product of RPL34-S. Figure 1 D is the electrophoresis result of the PCR amplification product of standard RPL34;

[0031] Figure 2 This is a diagram of the electrophoresis results of the plasmid enzyme digestion verification of the recombinant plasmid provided in the examples of this application, wherein: Figure 2 A is the electrophoresis result of the plasmid enzyme digestion verification of the recombinant plasmid of RPL34-H. Figure 2 B is the electrophoresis result of the enzyme digestion verification of the recombinant plasmid of RPL34-M. Figure 2 C is the electrophoresis result of the plasmid enzyme digestion verification of the recombinant plasmid of RPL34-S. Figure 2 D is the electrophoresis result of plasmid enzyme digestion verification of the recombinant plasmid of standard RPL34;

[0032] Figure 3 This is the electrophoresis result diagram of the optimization of the overexpression concentration and time of RPL34 provided in the examples of this application, wherein: Figure 3 A is the electrophoresis result of RPL34 at different overexpression concentrations. Figure 3 B is the electrophoresis results of RPL34 at different overexpression times;

[0033] Figure 4 This is a diagram of the WB electrophoresis results of screening siRNA-RPL34 provided in the examples of this application;

[0034] Figure 5 This is a diagram showing the infection results of porcine delta coronavirus mediated by overexpression of RPL34 in HIEC-6 cells provided in the examples of the present application, wherein: Figure 5 A is the statistical result of viral copy number detected by RT-qPCR at 12h and 24h respectively. Figure 5 B is the result of detecting virus titer using TCID50 at 12h and 24h respectively. Figure 5 C is the result of immunoblotting analysis to detect the effect of RPL34 overexpression on the level of porcine deltacoronavirus N protein at 12h and 24h, Figure 5 D is a staining image of infected cells at 24 h using flow cytometry and immunofluorescence to evaluate the effect of porcine delta coronavirus N antibody as the primary antibody and FITC-labeled goat anti-rabbit immunoglobulin (Cy3). Figure 5 E is a statistical diagram of the infection results of porcine delta coronavirus N antibody as the primary antibody and FITC-labeled goat anti-rabbit immunoglobulin (Cy3) at 24 hours using flow cytometry and immunofluorescence;

[0035] Figure 6 This is a diagram showing the infection results of porcine delta coronavirus mediated by knockdown of RPL34 in Caco-2 cells provided in the examples of the present application, wherein: Figure 6 A is the statistical result of viral copy number detected by RT-qPCR at 12h and 24h respectively. Figure 6 B is the result of detecting virus titer using TCID50 at 12h and 24h respectively. Figure 6 C is the result of immunoblotting analysis to detect the effect of RPL34 overexpression on the level of porcine deltacoronavirus N protein at 12h and 24h, Figure 6 D is a staining image of infected cells at 24 h using flow cytometry and immunofluorescence to evaluate the effect of porcine delta coronavirus N antibody as the primary antibody and FITC-labeled goat anti-rabbit immunoglobulin (Cy3). Figure 6 E is a statistical diagram of the infection results of porcine delta coronavirus N antibody as the primary antibody and FITC-labeled goat anti-rabbit immunoglobulin (Cy3) at 24 hours using flow cytometry and immunofluorescence;

[0036] Figure 7 This is a graph showing the results of infection with porcine delta coronavirus at different MOIs after overexpression of the RPL34 plasmid provided in the examples of this application in HIEC-6 cells, wherein: Figure 7 A is the immunoblotting test result of using RPL34 plasmid to overexpress 1μg, 2μg and 4μg of RPL34 and then infecting porcine delta coronavirus with 1MOI after 24 hours. Figure 7 B is the RT-qPCR test results of using RPL34 plasmid to overexpress 1μg, 2μg and 4μg of RPL34 and then infecting swine delta coronavirus with 1MOI after 24 hours. Figure 7 C is the immunoblotting result of using RPL34 plasmid to overexpress 2μg of RPL34 and then infecting porcine delta coronavirus with 1MOI after 24 hours. Figure 7 D is the RT-qPCR test result of using RPL34 plasmid to overexpress 2 μg of RPL34 and then infecting porcine delta coronavirus with 1 MOI after 24 hours;

[0037] Figure 8 This is a diagram showing the overexpression of RPL34 plasmids of different species in HIEC-6 cells provided in the examples of this application, wherein: Figure 8 A shows the results of immunoblotting for 24 hours after overexpression of mouse RPL34 plasmid using porcine delta coronavirus N antibody as the primary antibody. Figure 8 B is the RT-qPCR test results after overexpression of mouse RPL34 plasmid. Figure 8 C is the result of immunoblotting for 24 hours after overexpression of pig RPL34 plasmid using pig delta coronavirus N antibody as primary antibody. Figure 8 D is the RT-qPCR test results after overexpression of porcine RPL34 plasmid;

[0038] Figure 9 This is a diagram showing the results of the dose optimization of the inhibitor drug XL019 in inhibiting RPL34 provided in the examples of this application, wherein: Figure 9 A is the Western-Blot verification result of JAK inhibitor XL019 inhibiting RPL34. Figure 9 B is the RT-qPCR concentration verification of XL019 inhibition of RPL34. Figure 9 C is the cytotoxicity result of XL019 on Caco-2 cells;

[0039] Figure 10 This is a statistical graph of weight changes in KM mice challenged with poison after administration provided in the examples of the present application, wherein: Figure 10 A is attack 10 6 TCID50 Statistical chart of mouse weight changes, Figure 10 B is attack 10 7 TCID 50 Statistical chart of mouse weight changes, Figure 10 C is attack 10 8 TCID 50 Statistical graph of mouse weight changes;

[0040] Figure 11 This is a graph showing changes in intestinal viral load in KM mice challenged with a drug after administration provided in the examples of this application;

[0041] Figure 12 The pathological tissue images of the intestines of KM mice challenged with different drugs on the 10th day after the challenge provided in the examples of the present application;

[0042] Figure 13 Schematic diagram of the preparation process of siRNA-RPL34 provided in the examples of this application. DETAILED DESCRIPTION

[0043] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0044] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited number (fractional or integer) within the indicated range.

[0045] In this document, the terms including "including" and "comprising" mean "including but not limited to". Relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; wherein A and B can be singular or plural. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased on the market or prepared by existing methods.

[0046] The present application provides an embodiment of the present application providing an application of RPL34 protein as a biomarker of porcine deltacoronavirus, and the application includes: using overexpressed RPL34 protein as a biomarker of porcine deltacoronavirus infection in the body.

[0047] In some optional embodiments, the expression level of the overexpressed RPL34 protein is positively correlated with the degree of infection of the porcine delta coronavirus;

[0048] In these embodiments, the expression level of the overexpressed RPL34 protein can be positively correlated with the degree of infection of porcine delta coronavirus, and the direct association between the overexpressed RPL34 protein and the degree of infection of porcine delta coronavirus can be clarified to clarify the degree of direct association between the RPL34 protein and porcine delta coronavirus infection.

[0049] In some optional embodiments, the expression level of the overexpressed RPL34 protein is 5 times or more higher than the expression level of the normal RPL34 protein;

[0050] In these embodiments, the expression level of the overexpressed RPL34 protein can be 5 times or more higher than the expression level of the normal RPL34 protein, which can clarify the overexpression state of the expression level of the RPL34 protein, thereby further clarifying the degree of direct correlation between the RPL34 protein and porcine delta coronavirus infection.

[0051] Based on a general inventive concept, an embodiment of the present application provides a therapeutic drug for porcine deltacoronavirus, wherein the therapeutic drug includes an inhibitor that inhibits the RPL34 protein.

[0052] The therapeutic drug is based on the application of the above-mentioned RPL34 protein as a biomarker of porcine deltacoronavirus. The specific principle of the application of the RPL34 protein as a biomarker of porcine deltacoronavirus can be referred to the above-mentioned embodiments. Since the therapeutic drug adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0053] It should be noted that the inhibitor of RPL34 protein can be used in combination with a variety of other drugs.

[0054] In some optional embodiments, the inhibitor comprises the inhibitor drug XL019;

[0055] In some optional embodiments, the concentration of the inhibitor drug XL019 is ≥5 μM;

[0056] In these embodiments, the inhibitor may include the inhibitor drug XL019, and the concentration of the inhibitor drug XL019 may be ≥5 μM, which can prompt the inhibitor drug XL019 to effectively inhibit the expression of the RPL34 protein, thereby using the inhibitor drug XL019 to inhibit the expression of the RPL34 protein to inhibit the expression of porcine delta coronavirus, thereby achieving the treatment of porcine delta coronavirus infection.

[0057] In some optional embodiments, the inhibitor comprises siRNA-RPL34;

[0058] In some optional embodiments, the concentration of the siRNA-RPL34 is ≥10 μg / well;

[0059] In these embodiments, the inhibitor can include siRNA-RPL34, and the concentration of siRNA-RPL34 can be ≥10 μg / well, which can promote siRNA-RPL34 to effectively knock out or reduce the expression of RPL34 protein-related genes, and inhibit the expression of porcine delta coronavirus by using siRNA-RPL34 to inhibit the expression of RPL34 protein, so as to achieve the treatment of porcine delta coronavirus infection.

[0060] Figure 13 The following is a schematic diagram showing the process flow of the preparation method of siRNA-RPL34 provided in the examples of the present application;

[0061] like Figure 13 As shown, in some optional embodiments, the preparation method of the siRNA-RPL34 includes:

[0062] S1. PCR amplify the target gene fragment of the RPL34-related gene, and then recover it to obtain the amplified product;

[0063] S2. cloning the amplified product into an expression vector to obtain a recombinant plasmid;

[0064] S3. Enabling the recombinant plasmid to express siRNA-RPL34;

[0065] Wherein, the amplification primer set used for the PCR amplification includes a first primer set, a second primer set, a third primer set and a transcription primer set;

[0066] The forward primer of the first primer set is shown as SEQ ID NO.1, and the reverse primer of the first primer set is shown as SEQ ID NO.2;

[0067] The forward primer of the second primer set is shown as SEQ ID NO.3, and the reverse primer of the second primer set is shown as SEQ ID NO.4;

[0068] The forward primer of the third primer set is shown as SEQ ID NO.5, and the reverse primer of the third primer set is shown as SEQ ID NO.6;

[0069] The forward primer of the transcription primer set is shown in SEQ ID NO.7, and the reverse primer of the transcription primer set is shown in SEQ ID NO.8;

[0070] In these embodiments, multiple sets of PCR amplification primers are used to amplify RPL34-related genes to obtain sufficient amplification products, which are then used to construct recombinant plasmids, and finally, these recombinant plasmids are prompted to express siRNA-RPL34.

[0071] It should be noted that these target gene fragments can be RPL34 gene fragments of humans, pigs and mice.

[0072] Based on a general inventive concept, the present application provides a method for inhibiting porcine delta coronavirus infection for non-diagnostic and non-therapeutic purposes, the method comprising:

[0073] S1. Knock out the gene related to the expression of RPL34 protein in the body; and / or

[0074] S1. Use an inhibitor to inhibit RPL34 protein expression;

[0075] Wherein, when the method includes using an inhibitor to inhibit the expression of RPL34 protein, the inhibitor includes the inhibitor drug XL019 and / or siRNA-RPL34.

[0076] This method is based on the application of the above-mentioned RPL34 protein as a biomarker of porcine deltacoronavirus. The specific principle of the application of the RPL34 protein as a biomarker of porcine deltacoronavirus can be referred to the above-mentioned embodiments. Since this method adopts part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above-mentioned embodiments, which will not be repeated here.

[0077] The present application is further described below with reference to specific examples. Experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with industry standards; if there are no corresponding industry standards, they are measured in accordance with common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0078] Example 1

[0079] 1. Construction of recombinant expression plasmid:

[0080] The RPL34 genes of human, pig and mouse were amplified using the primers shown in the table. The results of nucleic acid electrophoresis showed a band of about 345 bp (e.g. Figure 1 This indicates that the RPL34 genes of different species were successfully amplified.

[0081] Table 1 List of amplification primers for each group

[0082]

[0083]

[0084] The PCR amplification products were recovered and cloned into pCAGGS and pLVX-TRE3G vectors, respectively, to construct recombinant plasmids pCAGGS-RPL34-H, pCAGGS-RPL34-M, pCAGGS-RPL34-S, and pLVX-TRE3G-RPL34-H, which were double-digested with restriction endonucleases EcoRI and XhoI, and EcoRI and BamHI, as shown in Figure 2 . Figure 2 As shown, a band of about 345 bp can be seen after electrophoresis detection, which indicates that each recombinant plasmid was successfully constructed.

[0085] 2. Optimization of recombinant plasmid transfection dose and time:

[0086] Transfect 2 μg / well of the same concentration of pCAGGS-RPL34-H into HIEC-6 cells, and collect the cells at different culture time periods to detect the expression effect of the recombinant plasmid in the cells, such as Figure 3 As shown in Figure 2, the recombinant plasmid can successfully express an 18kDa protein in cells. It can be seen that the protein expression level reaches the highest at 48h (as shown in Figure 2). Figure 3Then, HIEC-6 cells were transfected with pCAGGS-RPL34-H at different concentrations of 1 μg / well, 2 μg / well, and 4 μg / well. The cells were harvested after 48 hours. The expression level of RPL34 was the highest at a concentration of 2 μg / well (as shown in Figure 2A). Figure 3 Therefore, the transfection time and dosage for subsequent operations were selected as 48h and 2μg / well.

[0087] 3. RPL34 gene siRNA screening:

[0088] Three siRNAs were designed and synthesized targeting the coding region of the human RPL34 gene, named siRNA-RPL34 No. 1, siRNA-RPL34 No. 2, and siRNA-RPL34 No. 3. Then, the three siRNA-RPL34 were transfected into Caco-2 cells at 50nM / well. After culturing for 36 hours, the cell proteins were collected for WB detection. The results are as follows Figure 4 As shown, it was found that these siRNA-RPL34s all successfully knocked down the expression of RPL34 in Caco-2, and the effect of siRNA-RPL34 No. 2 was the most obvious.

[0089] 4. Overexpression of RPL34 promotes porcine deltacoronavirus infection:

[0090] After overexpression of RPL34 protein for 48 hours, HIEC-6 cells were infected with porcine delta coronavirus at 1 MOI. To evaluate the functional impact of RPL34 overexpression, a variety of experimental techniques including Western blot analysis, RT-qPCR, TCID50 detection, flow cytometry and immunofluorescence were used to detect porcine delta coronavirus infection in HIEC-6 cells. The primary antibody used in these techniques was a polyclonal antibody against the N protein of porcine delta coronavirus, which specifically detected a 42 kDa band. Figure 5 As shown in Figure 2, Western Blot analysis showed that after RPL34 overexpression, the expression of target bands and N protein increased significantly (e.g. Figure 5 As shown in A). mRNA detection showed significant differences in N protein expression at the two time points (as shown in Figure 5 In addition, TCID50 assay showed that the virus TCID50 in the supernatant decreased at 12h and 24h after infection (as shown in Figure 2B). Figure 5 C).

[0091] Flow cytometry analysis using FITC-coupled anti-swine deltacoronavirus N antibody showed that the proportion of positive cells in HIEC-6 cells overexpressing RPL34 was lower (e.g. Figure 5Finally, immunofluorescence results using porcine deltacoronavirus N antibody as the primary antibody showed that the specific fluorescence of N protein in cells overexpressing RPL34 was significantly increased compared with that in untransfected cells (as shown in Figure 2). Figure 5 These findings indicate that overexpression of RPL34 can effectively enhance the infection process of porcine deltacoronavirus.

[0092] 5. Research on knocking down RPL34 to inhibit porcine delta coronavirus infection:

[0093] RPL34 was knocked down in Caco-2 cells, and a variety of experimental techniques including Western blot analysis, RT-qPCR, TCID50, flow cytometry and immunofluorescence were used for detection. The primary antibodies used in these detection techniques can specifically target the porcine delta coronavirus N protein and can detect a band with a molecular weight of 42 kDa. In addition, Western blot analysis results showed that after RPL34 inhibition, the expression of porcine delta coronavirus N protein decreased (such as Figure 6 In addition, the results of RT-qPCR analysis showed that there were significant differences in the expression of N protein at the two time points (as shown in Figure 6 In addition, the TCID50 test results showed that the virus TCID50 in the supernatant decreased after 12h and 24h of infection (as shown in Figure 2). Figure 6 C)

[0094] Flow cytometric analysis using FITC-conjugated anti-porcine deltacoronavirus N antibody showed that the percentage of positive cells was lower in Caco-2 cells with RPL34 inhibition compared with that in the siRNA-NC group (Fig. Figure 6 Finally, immunofluorescence results using porcine deltacoronavirus N antibody as the primary antibody showed that the specific fluorescence of RPL34 protein in the inhibitory cells was reduced compared with that in untransfected cells (as shown in Figure 6 E).

[0095] These results indicate that inhibition of RPL34 can effectively reduce porcine deltacoronavirus infection.

[0096] 6. Evaluating the Dose-dependence of Porcine Delta Coronavirus on RPL34

[0097] In order to evaluate the effect of different concentrations of RPL34 on the infection of porcine delta coronavirus in HIEC-6 cells, 1μg, 2μg or 4μg of RPL34 plasmid were transfected into the cells, and then the cells were infected with porcine delta coronavirus. The infection effect of the cells was determined by Western-Blot and RT-qPCR. The results showed that compared with the overexpression of 1μg and 2μg of RPL34, the overexpression of 4μg of RPL34 promoted the infection of porcine delta coronavirus to a greater extent (such as Figure 7 A and Figure 7 (As shown in Figure B). The inhibitory effect of RPL34 on porcine deltacoronavirus is dose-dependent, with the higher the concentration, the more pronounced the promoting effect.

[0098] To investigate the potential effects of RPL34 on porcine delta coronavirus infection at different MOIs, HIEC-6 cells were transfected with 2 μg of RPL34 and subsequently infected with porcine delta coronavirus at MOIs of 0.1, 1, and 2. Western blot and RT-qPCR were then used to assess infection. The results showed that overexpression of 2 μg of RPL34 at an MOI of 0.1 significantly enhanced the effect of porcine delta coronavirus infection compared to MOIs of 1 and 2 (e.g., Figure 7 C and Figure 7 D). These results indicate that RPL34 has a better promoting effect on porcine delta coronavirus at a lower MOI value.

[0099] 7. Overexpression of mouse and porcine RPL34 promotes the replication of porcine deltacoronavirus in HIEC-6 cells:

[0100] After 48 hours of overexpression of mouse and porcine RPL34, HIEC-6 cells were infected with 1 MOI of porcine delta coronavirus. In order to evaluate the functional effects of RPL34 overexpression, Western blot analysis and RT-qPCR were used for detection. The primary antibody used in this application is a polyclonal antibody against the N protein of porcine delta coronavirus, which specifically detects a 42 kDa band. Western blot analysis results showed that after RPL34 overexpression, the expression of the target band and N protein increased significantly (such as Figure 8 A and Figure 8 C). mRNA detection showed significant differences in N protein expression at the two time points (as shown in Figure 8 B and Figure 8 D). This indicates that RPL34 from different species can promote infection by porcine delta coronavirus.

[0101] 8. Optimization of drug dosage and duration for targeted inhibition of RPL34:

[0102] The inhibitor drug XL019 was added to Caco-2 cells at 2μM, 5μM and 8μM, and then infected with porcine delta coronavirus. The cells were subjected to WB and mRNA detection 24 hours after infection. The results showed that XL019 above 5μM could significantly inhibit the expression of RPL34 and porcine delta coronavirus N protein (such as Figure 9 A and Figure 9 The CCK8 assay was then used to test drug cytotoxicity over a 24-hour period. The results showed significant cytotoxicity at a dose of 8 μM. Therefore, a 5 μM dose was selected for subsequent drug efficacy experiments.

[0103] 9. Body weight changes of Kunming (KM) mice challenged with poison after drug administration:

[0104] XL019 was administered to KM mice by gavage at a dose of 500 μg / mouse. After 7 days of administration, KM mice were given 10 6 , 10 7 , 10 8 TCID 50 The pigs were challenged with pig Delta coronavirus by gavage and grouped as shown in Table 2. The body weight was recorded every day. 6 TCID 50 There was no significant difference in the weight change trend between the drug-treated group and the non-drug-treated group; 7 , 10 8 TCID 50 The body weight of the mice in both the drug-treated and non-drug-treated groups decreased significantly on the second day, which indicates that the drug has a certain protective effect on the intestinal tract of KM mice (such as Figure 10 shown).

[0105] Table 2 Distribution of JAK-administered mice

[0106] Group Dosage Virus types Challenge dose <![CDATA[10 6 TCID50]]> - porcine deltacoronavirus <![CDATA[10 6 TCID 50 / mL]]> <![CDATA[XL019-10 6 TCID50]]> 500 μg porcine deltacoronavirus <![CDATA[10 6 TCID 50 / mL]]> <![CDATA[10 7 TCID50]]> - porcine deltacoronavirus <![CDATA[10 7 TCID 50 / mL]]> <![CDATA[XL019-10 7 TCID50]]> 500 μg porcine deltacoronavirus <![CDATA[10 7 TCID 50 / mL]]> <![CDATA[10 8 TCID50]]> - porcine deltacoronavirus <![CDATA[10 8 TCID 50 / mL]]> <![CDATA[XL019-10 8 TCID50]]> 500 μg porcine deltacoronavirus <![CDATA[10 8 TCID 50 / mL]]> PBS - PBS -

[0107] 9. Effect of XL019 on the viral load in the intestinal tissue of KM mice challenged with the virus

[0108] On the 10th day after the challenge, intestinal tissue samples of mice were collected and the viral load of the porcine delta coronavirus N protein in the jejunum was detected. It was found that at different challenge doses, XL019 had a significant inhibitory effect on the porcine delta coronavirus infection load. However, the inhibitory effect gradually deteriorated with the increase of the viral load (such as Figure 11 shown).

[0109] 10. Intestinal histopathological observation:

[0110] Then, 10 samples collected on the 10th day after infection were 8Pathological sections of the intestinal tissues of mice in the TCID50 group, XL019 inhibitor group, and PBS group were examined. After drug administration, the intestinal tissues of the small intestine that was attacked by the poison showed a large amount of autolysis of the villi tips (yellow arrows in the figure); the intestinal glands in the lamina propria were abundant, densely arranged, and short tubular, with few goblet cells; the muscle layer structure was clear, the muscle cells were arranged regularly, and no other obvious abnormalities were observed. After drug administration, the intestinal tissues of the small intestine showed a large amount of autolysis of the villi tips (yellow arrows in the figure); the lamina propria had focal edema (purple arrows in the figure), the connective tissue was loosely arranged, the blood vessels were dilated, and there was a very small amount of scattered lymphocyte infiltration (blue arrows in the figure); the muscle layer structure was clear, the muscle cells were arranged regularly, and no other obvious abnormalities were observed; in addition, no lesions were observed in the PBS group (such as Figure 12 This also proves that XL019 can inhibit the infection of porcine delta coronavirus.

[0111] In summary, the application of an RPL34 protein as a biomarker of porcine delta coronavirus provided in the embodiment of the present application, through the above experiments, it was found that overexpression of RPL34 protein has a promoting effect on the infection process of porcine delta coronavirus, and the expression level of RPL34 protein was significantly increased in cells infected with porcine delta coronavirus, indicating that the expression of RPL34 protein can be used as a type of biomarker for porcine delta coronavirus infection of the body. In addition, the use of the inhibitor drug XL019 and siRNA-RPL34 that can knock down RPL34 to inhibit the expression of RPL34 can inhibit the infection level of porcine delta coronavirus, thereby filling the gap between the association between RPL34 protein and porcine delta coronavirus infection.

[0112] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but rather is intended to conform to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. An inhibitor of RPL34 protein is used in the preparation of a therapeutic drug for treating porcine delta coronavirus infection, characterized in that: The therapeutic drug is the inhibitor drug XL019.

2. The use according to claim 1, characterized in that The concentration of the inhibitor drug XL019 is ≥5 μM.

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