Application of silencing ECM1 gene in resistance to PRRSV infection

Abstract

The present invention provides the use of silencing the ECM1 gene in resisting PRRSV infection, belonging to the field of medical technology. The present invention provides the use of a reagent that affects the expression level of the ECM1 gene in the preparation of a medicament for regulating PRRSV infection in animals, as well as an siRNA that inhibits porcine reproductive and respiratory syndrome virus infection in animals. After the present invention used two siRNAs to interfere with ZMAC-4 cells respectively, the expression of ECM1 and PRRSV-ORF6 in the cells transfected with siRNA was significantly reduced, indicating that interfering with the expression of the ECM1 gene has an inhibitory effect on PRRSV infection of ZMAC-4 cells.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to application of silencing ECM1 gene in resisting PRRSV infection. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease of pigs caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It can cause reproductive disorders in pregnant sows and respiratory syndrome in weaned piglets, impacting the development of pig farming. PRRSV infection can be divided into three distinct phases: acute, persistent, and resolved. During the acute phase, the lungs are the primary organ attacked by PRRSV.

[0003] The extracellular matrix (ECM) is a complex network of proteins and proteoglycans composed of a large number of proteins, including collagen, glycoproteins, and ECM-related proteins, which support, structure, and regulate cell function. Remodeling or accumulation of the ECM can lead to lung damage and thus cause chronic lung diseases such as pulmonary fibrosis. Therefore, the ECM may play a role in PRRSV infection, and the glycoprotein encoded by ECM1 is an important component of the ECM. The ECM1 protein can interact with other proteins such as collagen and fibronectin to form a structural network that gives tissue strength and elasticity. In addition, the ECM1 protein is also associated with disease processes such as tumor development and metastasis, and fibrosis. Therefore, by studying the role of ECM1 in PRRSV infection and exploring therapeutic targets and pathogenesis of PRRSV infection, it will help promote the development of the pig industry. Summary of the Invention

[0004] The present invention provides an application of silencing the ECM1 gene in resisting PRRSV infection, and finds that interfering with ECM1 expression can inhibit the invasion and replication of PRRSV into host cells.

[0005] The present invention provides the use of a reagent that affects the expression level of the ECM1 gene in preparing a medicament for regulating and controlling animals infected with porcine reproductive and respiratory syndrome virus.

[0006] In one embodiment of the present invention, the reagent includes a reagent that inhibits the expression of the ECM1 gene.

[0007] In one embodiment of the present invention, the agent for inhibiting the expression of the ECM1 gene comprises siRNA.

[0008] In one embodiment of the present invention, the siRNA includes ECM1-sus-668 and ECM1-sus-911;

[0009] Wherein, ECM1-sus-668 comprises a sense strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2;

[0010] ECM1-sus-911 includes a sense strand represented by the nucleotide sequence of SEQ ID No. 3 and an antisense strand represented by SEQ ID No. 4.

[0011] The present invention also provides an siRNA for inhibiting porcine reproductive and respiratory syndrome virus infection in animals, wherein the siRNA comprises ECM1-sus-668 and ECM1-sus-911;

[0012] Wherein, ECM1-sus-668 comprises a sense strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2;

[0013] ECM1-sus-911 includes a sense strand represented by the nucleotide sequence of SEQ ID No. 3 and an antisense strand represented by SEQ ID No. 4.

[0014] In one embodiment of the present invention, the animal comprises a pig.

[0015] The present invention also provides the application of the above siRNA in the study of the mechanism of action of porcine reproductive and respiratory syndrome.

[0016] The present invention also provides the use of the above siRNA in preparing a drug for preventing and / or treating porcine reproductive and respiratory syndrome.

[0017] The present invention also provides the application of the above siRNA in pig breeding and production.

[0018] Beneficial Effects: The present invention provides the use of agents that influence ECM1 gene expression in the preparation of pharmaceutical agents for regulating PRRSV infection in animals. In the examples, five splice isoforms of ECM1 were cloned from normal porcine lung tissue. Two siRNAs, ECM1-sus-668 and ECM1-sus-911, were used. The siRNA (ECM1-sus-668) targeted a wider range of transcripts. ZMAC-4 cells were subjected to siRNA interference with siRNA-668 and siRNA-911 for 24 hours, followed by PRRSV (XH-GD) inoculation for 24 hours, resulting in a viral titer of 106.7. Expression of ECM1 and PRRSV-ORF6 was significantly decreased in cells transfected with the siRNAs, indicating that interference with ECM1 gene expression inhibits PRRSV infection in ZMAC-4 cells. Mechanistic studies have revealed that ECM1 may influence PRRSV invasion and replication in host cells by affecting the ITGB3-AKT / FAK pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a graph showing the relative expression of ECM1 in various tissues;

[0020] Figure 2 is a diagram of coding sequence differences among the five transcripts;

[0021] Figure 3 It is the alignment diagram of the transcript with the corresponding transcript sequence and amino acid sequence in the database;

[0022] Figure 4 The graph shows the results of siRNA (ECM1-sus-668) and siRNA (ECM1-sus-911) inhibiting the expression of ECM1 and ORF-6 mRNA;

[0023] Figure 5 Graph showing the effect of siRNA (ECM1-sus-911) on ITGB3, AKT2 and FAK gene expression. DETAILED DESCRIPTION

[0024] The present invention provides the use of a reagent that affects the expression level of the ECM1 gene in preparing a medicament for regulating animals infected with porcine reproductive and respiratory syndrome virus (PRRSV).

[0025] In one embodiment of the present invention, the reagent includes an agent that inhibits ECM1 gene expression. In one embodiment, the expression of the ECM1 gene is interfered with using siRNA. In one embodiment of the present invention, when the animal is a pig, the siRNA includes ECM1-sus-668 and ECM1-sus-911; wherein ECM1-sus-668 includes the nucleotide sequence of the sense strand shown in SEQ ID No. 1 and the antisense strand shown in SEQ ID No. 2;

[0026] ECM1-sus-911 includes a sense strand represented by the nucleotide sequence of SEQ ID No. 3 and an antisense strand represented by SEQ ID No. 4.

[0027] The ECM1 gene described herein includes various transcripts related to the ECM1 gene. Five transcripts were verified in the Examples, with accession numbers PP885370, PP885371, PP885372, PP885373, and PP885374 in GenBank (http: / / www.ncbi.nlm.nih.gov / ). The results of the Examples indicate that siRNA (ECM1-sus-668) and siRNA (ECM1-sus-911) inhibited ECM1 transcript expression, with siRNA (ECM1-sus-668) targeting a wider range of transcripts. Cells transfected with these two siRNAs showed a significant decrease in ECM1 expression. RT-qPCR was used to detect intracellular RRRSV-ORF6. The results showed that both siRNA (ECM1-sus-668) and siRNA (ECM1-sus-911) interference significantly inhibited PRRSV-ORF6 mRNA expression, indicating that interfering with the expression of the ECM1 gene had an inhibitory effect on PRRSV infection of ZMAC-4 cells.

[0028] ECM1-Sus-668:

[0029] SEQ ID No.1: 5'-GCUGUCACAACUACAUACATT-3';

[0030] SEQ ID No.2: 5'-UGUAUGUAGUUGUGACAGCTT-3';

[0031] ECM1-Sus-911:

[0032] SEQ ID No.3: 5'-CACCCACACUGGACAAUAUTT-3';

[0033] SEQ ID No. 4: 5'-AUAUUGUCCAGUGUGGGUGTT-3'.

[0034] The present invention also provides an siRNA for inhibiting porcine reproductive and respiratory syndrome virus infection in animals, wherein the siRNA comprises ECM1-sus-668 and ECM1-sus-911;

[0035] Wherein, ECM1-sus-668 comprises a sense strand as shown in SEQ ID No. 1 and an antisense strand as shown in SEQ ID No. 2;

[0036] ECM1-sus-911 includes a sense strand represented by the nucleotide sequence of SEQ ID No. 3 and an antisense strand represented by SEQ ID No. 4.

[0037] In one embodiment of the present invention, the animal comprises a pig.

[0038] The present invention also provides the application of the above siRNA in the study of the mechanism of action of porcine reproductive and respiratory syndrome.

[0039] The present invention also provides the use of the above siRNA in preparing a drug for preventing and / or treating porcine reproductive and respiratory syndrome.

[0040] The present invention also provides the application of the above siRNA in pig breeding and production.

[0041] To further illustrate the present invention, the application of the silencing ECM1 gene provided by the present invention in resisting PRRSV infection is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1 Expression differences of ECM1 gene in different tissues

[0043] Three 30-day-old Large White pigs (purchased from Zhongshan Baishi Pig Farm) were slaughtered and tissue samples including heart, liver, spleen, lung, kidney, duodenum, and longissimus dorsi muscle were isolated from each pig.

[0044] Total RNA from each tissue sample was extracted using the Trizol (Invitrogen) method, and RNA concentration was determined using a micro-nucleic acid protein analyzer (Quawell Q500). RNA integrity was checked by 1% agarose gel electrophoresis. A reverse transcription first-strand cDNA synthesis kit ( IIIAll-in-one RT SuperMix Perfect For qPCR), according to the kit instructions, 1 μg of total RNA was used for first-strand cDNA synthesis.

[0045] The expression profile of ECM1 gene in different tissues of pigs was analyzed by fluorescence quantitative PCR. Figure 1 As shown, ECM1 gene is expressed in heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, leg muscle, pancreas, fat and other tissues and organs. GAPDH is used as a housekeeping gene by 2 -ΔΔCt The relative expression levels of ECM1 in various tissues were calculated. The results showed that ECM1 mRNA expression was highest in adipose tissue, followed by liver tissue. ECM1 mRNA expression was relatively lowest in leg muscle.

[0046] The ECM1 qPCR primer sequences are as follows:

[0047] Q-ECM1-F (SEQ ID No.5): 5'-AGTCCCAGAGGGAGTTTCCA-3';

[0048] Q-ECM1-R (SEQ ID No. 6): 5'-TGCAGCATAGCCAGCTTCTT-3';

[0049] GAPDH qPCR primer sequences are as follows:

[0050] GAPHD-F (SEQ ID No. 7): 5'-GTCGGAGTGAACGGATTTGG-3';

[0051] GAPHD-R (SEQ ID No. 8): 5'-TTGACTGTGCCGTGGAACTT-3'.

[0052] The qPCR reaction system consisted of 10 μL ChamQ Universal SYBR qPCR Master Mix (Novozymes, Nanjing, China), 0.5 μL upstream primer, 0.5 μL downstream primer, and 1 μL cDNA. The PCR reaction program was as follows: step 1, 95°C for 30 sec; step 2, 95°C for 10 sec, and 60°C for 30 sec; step 3, the instrument default melting curve acquisition program was used.

[0053] Example 2 Cloning of ECM1 transcripts

[0054] Primers were designed to amplify the full-length coding sequence to obtain different alternative splicing variants of ECM1. 1 μL of Large White pig tissue sample cDNA was used as the template for ECM1 gene amplification, and ECM1 gene-specific primers were used.

[0055] ECM1-specific primers are as follows:

[0056] ECM1-F (SEQ ID No. 9): 5'-ATGGGGACCACGTCCAGAGCA-3';

[0057] ECM1-R (SEQ ID No. 10): 5'-TCACTCTTCCTTGGGCTCAAGG-3'.

[0058] The PCR reaction system was as follows: cDNA 1 μL, RNase-free double-distilled water 32 μL, upstream primer 2 μL, downstream primer 2 μL, 10 μL of FastPfu Buffer and 4 μL of 2.5 mM dNTPs were used; the PCR reaction program was as follows: 95°C for 2 min; 95°C for 20 s, 58°C for 20 s, and 72°C for 1 min, 35 cycles; and 72°C for 5 min.

[0059] Nested PCR ECM1-specific primers are as follows:

[0060] N-ECM1-F (SEQ ID No. 11): 5'-TTCTTGTAGTCCCAGAGGGAGTT-3';

[0061] N-ECM1-R (SEQ ID No. 12): 5'-AGCAGTTGACCTGTTCATCCCC-3'.

[0062] The reaction system of nested PCR was as follows: 1 μL of PCR product, 32 μL of RNase-free double-distilled water, 2 μL of upstream primer, 2 μL of downstream primer, 10 μL of FastPfu Buffer and 4 μL of 2.5 mM dNTPs were used; the nested PCR reaction program was: 95°C for 2 min; 95°C for 20 s, 58°C for 20 s, 72°C for 1 min, 35 cycles; and 72°C for 5 min.

[0063] according to 5' / 3' RACE first-strand cDNA synthesis was performed according to the RACE 5' / 3' Kit User Manual (TaKaRa, Dalian, China).

[0064] The reaction procedure is as follows: 42℃ for 90min, 70℃ for 10min. Add 10μL Tricine-EDTA Buffer to dilute the first-strand cDNA and store at -20℃. RACE 5' / 3' Kit User Manual (TaKaRa, Dalian, China) Rapid cDNA End Amplification Protocol: 50 μL PCR reaction volume: 15.5 μL PCR-Grade water, 25.0 μL 2X SeqAmp Buffer, 1.0 μL SeqAmp DNA Polymerase, 2.5 μL 5'- or 3'-RACE-Ready cDNA, 5 μL 10X UPM, and 1 μL 5'- or 3' GSP (10 μM). PCR reaction conditions: 94°C for 30 seconds, 68°C for 30 seconds, and 72°C for 4 minutes, for 30 cycles.

[0065] UPM long (SEQ ID No. 13): 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3';

[0066] GSP3 (SEQ ID No. 14): 5'-ACCCACCGCCACTTGTGTTGCCACCA-3';

[0067] GSP5 (SEQ ID No. 15): 5'-AGGGCTCTGCTGAGAAGTGTCAGGGGGA-3'.

[0068] 5'RACE, 3'RACE, and coding region PCR products were separated by 1% agarose gel electrophoresis and purified using a gel extraction kit (TAKARA, Dalian, China). The purified product was reacted with pEASY-Blunt Cloning Vector (TRANS, Beijing, China) at a vector-to-fragment molar ratio of 1:7 at room temperature (20°C-37°C) for 5 minutes. The reaction product was transferred into 50 μL of Trans1-T1 competent cells and gently flicked to mix. The cells were then incubated on ice for 20-30 minutes. Heat shock was performed in a 42°C water bath for 30 seconds and immediately placed on ice for 2 minutes. After adding 250 μL of SOC medium and incubating at 37°C (200 rpm) with shaking for 1 hour, the bacterial suspension was evenly plated onto LB solid medium containing ampicillin, IPTG, and X-gal. After incubation at 37°C for 14 hours, positive single colonies were selected and incubated in ampicillin liquid medium with shaking at 37°C for 8 hours. Then, the cultured bacterial fluid was detected by PCR, and five ECM1 alternative splicing variants were detected in pig lung tissue by Sanger sequencing. The five transcripts corresponding to the pig ECM1 annotated in the NCBI database were extracellular matrix protein 1isoform X2 (XM_021089906.1), extracellular matrix protein 1isoform X4 (XM_021089908.1), extracellular matrix protein 1isoform X5 (XM_021089909.1), extracellular matrix protein 1isoform X6 (XM_021089910.1), and extracellular matrix protein 1isoform X7 (XM_021089911.1), which were named ecm1-2, ecm1-4, ecm1-5, ecm1-6, and ecm1-7 according to the corresponding transcript sequences in NCBI, respectively. Their sequences have been deposited in GenBank ( http: / / www.ncbi.nlm.nih.gov / ) with accession numbers (PP885370 ), (PP885371 ), (PP885372 ), (PP885373 ), and (PP885374 ), respectively.

[0069] By comparing the sequences of these five variants, the sequence differences between them were analyzed. Figure 2As shown, the full-length coding sequence of ECM1-2 is 1695 bp. Compared with ECM1-2, the full-length coding sequence of ECM1-4 is 1641 bp, with a 54 bp deletion between 391-444 bp. The full-length coding sequence of ECM1-5 is 1626 bp, and compared with ECM1-2, ECM1-5 has a 69 bp deletion between 391-459 bp. The full-length coding sequence of ECM1-6 is 1320 bp, and compared with ECM1-2, ECM1-6 has a 375 bp deletion between 789-1163 bp. The full-length coding sequence of ECM1-7 is 1251 bp, and compared with ECM1-2, ECM1-7 has a 69 bp deletion between 391-459 bp and a 375 bp deletion between 789-1163 bp.

[0070] Example 3 Sequence Analysis of ECM1 Gene

[0071] Open reading frames were predicted using NCBIORF Finder, and protein physicochemical properties were predicted using Expasy software. Signal peptides were predicted using the online prediction software NovoPro. Transmembrane domains were predicted using the online software TMHMM. Subcellular localization of target genes was predicted using the online software WoLFPSORT.

[0072] The results are as follows Figure 3 As shown, the five transcripts of the ECM1 gene differ from the corresponding transcripts in the NCBI database by only one base sequence difference, with a base sequence similarity of 99%, but they encode the same protein. They also have the same 124bp 5' untranslated region (UTR) and 409bp 3' untranslated region (UTR).

[0073] Software predicted that ECM1s share a common signal peptide type: SP (Sec / SPI); the signal peptide sequence is SEQ ID No. 16 (MGTTSRAALVLAYLAVASVVPEGVS), indicating that ECM1 is a secreted protein. WoLF PSORT online software predicted that ECM1s are located extracellularly. Furthermore, the software predicted that ECM1s lack a transmembrane domain, further confirming that porcine ECM1 is a secreted protein.

[0074] Example 4: Inhibitory effect of silencing ECM1 differential transcripts on PRRSV infection in ZMAC-4 cells

[0075] Cell culture system: 10% FBS + 1640 medium.

[0076] siRNA (ECM1-sus-668) and siRNA (ECM1-sus-911) were used to inhibit ECM1 transcript expression, among which siRNA (ECM1-sus-668) could target a wider range of transcripts.

[0077] ZMAC-4 cells were seeded in 6-well plates and transfected when cells reached 70% confluence according to the manufacturer's instructions for Lipofectamine 3000. Lipofectamine 3000 reagent was diluted in Opti-MEM medium. siRNA was diluted in Opti-MEM medium and mixed thoroughly to prepare a premix. The premix was added to the diluted Lipofectamine 3000 reagent at a ratio of 1:1, diluted for 10-15 minutes, and the mixed solution was added to the cells. Cells were transfected with 50 pM siRNA-668, siRNA-911, or the negative control siRNA-NC. siRNA interference was performed for 24 hours, followed by PRRSV (XH-GD) inoculation for 24 hours, resulting in a viral titer of 106.7. The XH-GD strain was donated by Foshan University of Science and Technology and published in the literature [Cao ZX, Jiao PR, Huang YM, et al. Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China [J]. Acta Vet Hung, 2012, 60(1): 157-164.].

[0078] Cell samples were collected and total RNA was extracted and reverse transcribed to synthesize cDNA as described in Example 1. RT-qPCR was performed to analyze the effects of siRNA. NC-PRRSV was the negative control siRNA-NC treatment group; ECM1-sus-668-PRRSV was the siRNA (ECM1-sus-668) treatment group; and ECM1-sus-911-PRRSV was the siRNA (ECM1-sus-911) treatment group. The results are shown in Figure 1. Figure 4 As shown, ECM1 expression was significantly decreased in cells transfected with siRNA. RT-qPCR was used to detect intracellular RRRSV-ORF6. The results showed that both siRNA (ECM1-sus-668) and siRNA (ECM1-sus-911) significantly inhibited PRRSV-ORF6 mRNA expression. These results indicate that interfering with ECM1 gene expression inhibits PRRSV infection of ZMAC-4 cells.

[0079] The ITGB3-AKT / FAK pathway was detected by RT-qPCR. Figure 5 As shown in the figure, compared with the control group, the expression of ITGB3, AKT2, and FAK mRNA was significantly downregulated after siRNA (ECM1-sus-911) treatment, indicating that ECM1 may affect the invasion and replication of PRSSV in host cells by affecting the ITGB3-AKT / FAK pathway.

[0080] The siRNA (ECM1-sus-668) sequence is as follows:

[0081] SEQ ID No.1: 5'-GCUGUCACAACUACAUACATT-3';

[0082] SEQ ID No.2: 5'-UGUAUGUAGUUGUGACAGCTT-3';

[0083] The siRNA (ECM1-sus-911) sequence is as follows:

[0084] SEQ ID No.3: 5'-CACCCACACUGGACAAUAUTT-3';

[0085] SEQ ID No.4: 5'-AUAUUGUCCAGUGUGGGUGTT-3';

[0086] The siRNA (NC) sequence is as follows:

[0087] SEQ ID No.25: 5'-UUCUCCGAACGUGUCACGUTT-3';

[0088] SEQ ID No. 26: 5'-ACGUGACACGUUCGGAGAATT-3'.

[0089] PRRSV-ORF6 qPCR primer sequences are as follows:

[0090] PRRSV-ORF6-F (SEQ ID No. 17): 5'-TTTGACTGGGCAGTGGAGAC-3';

[0091] PRRSV-ORF6-R (SEQ ID No. 18): 5'-AGGAAATGGCTGGTGGTGAG-3';

[0092] ITGB3 qPCR primer sequences are as follows:

[0093] ITGB3-F (SEQ ID No. 19): 5'-GAGTTCCCCATCAGTGAGGC-3';

[0094] ITGB3-R (SEQ ID No. 20): 5'-CTCTTCGAATCATCTGGCCG-3';

[0095] The AKT2 qPCR primer sequences are as follows:

[0096] AKT2-F (SEQ ID No. 21): 5'-GGGTTCTCCAGAACACCAGG-3';

[0097] AKT2-R (SEQ ID No. 22): 5'-CAGGTGGAAAAACAGCTCGC-3';

[0098] The FAK qPCR primer sequences are as follows:

[0099] FAK-F (SEQ ID No. 23): 5'-TGCTGGACCTCTGAACTAATATGAC-3';

[0100] FAK-R (SEQ ID No. 24): 5'-GTGCCCAGGTGAGTCTTGG-3'.

[0101] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. The use of siRNA in the preparation of a medicament for regulating porcine reproductive and respiratory syndrome virus infection in animals, characterized in that: The siRNA is ECM1-sus-911; The nucleotide sequence of ECM1-sus-911 is the sense strand shown in SEQ ID No. 3 and the antisense strand shown in SEQ ID No.

4.

2. An siRNA for inhibiting porcine reproductive and respiratory syndrome virus infection in pigs, characterized in that: The siRNA is ECM1-sus-911; The nucleotide sequence of ECM1-sus-911 is the sense strand shown in SEQ ID No. 3 and the antisense strand shown in SEQ ID No.

4.

3. Use of the siRNA according to claim 2 in the preparation of a drug for treating porcine reproductive and respiratory syndrome.

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