Application of DEFA1 in COVID-19 Infection

Abstract

The present invention discloses a method for using a biotherapy for novel coronavirus (SARS-CoV-2) infection, wherein the protein is DEFA1 (human defensin α1, or human neutrophil peptide, HNP-1). The reagents include but are not limited to: 1) DEFA1 recombinant protein; 2) a vector containing a DEFA1 encoding gene; 3) a reagent for detecting RNA expression of DEFA1. The experimental research results of the present invention provide a new biotherapy method for novel coronavirus infection, and provide a new drug target for treating respiratory tract lesions caused by novel coronavirus infection.

Description

Technical Field

[0001] The present invention relates to the field of medical biotherapy technology, and specifically, to a diagnostic and therapeutic application of DEFA1 in novel coronavirus infection. Background Art

[0002] At present, the main drugs for treating COVID-19 include neutralizing antibodies, traditional Chinese medicine, and small molecule antiviral drugs. However, there is still a lack of efficient treatment methods for its treatment, and it is impossible to completely eliminate the virus from the root. Therefore, finding antiviral biomolecules that specifically target SARS-CoV-2 infection is an urgent problem that needs to be solved. Summary of the invention

[0003] The study found that DEFA1 is a type of polypeptide protein, mainly produced by neutrophils, and is an important effector molecule of innate immunity. The results also showed that DEFA1 plays a key role in phagocyte-mediated autoimmune protection. As a broad-spectrum antimicrobial protein, the expression of DEFA1 may be a defensive response of the body against viral infection.

[0004] Specifically, current studies have found that high levels of DEFA1 can be detected in the serum of patients infected with the new coronavirus, and the expression of DEFA1 in the patients' mononuclear cells is also increased. Studies have shown that DEFA1 can inhibit a variety of viral infections, including human immunodeficiency virus (HIV), influenza virus, adenovirus, etc. DEFA1 can simultaneously inhibit the invasion and replication of HIV, and can also inhibit the replication of influenza virus and the synthesis of viral proteins in vitro. In addition, DEFA1 can significantly reduce the infection of adenovirus type 5 in 293 cells, and high levels of DEFA1 can also be detected in the nasal secretions of children infected with adenovirus.

[0005] Based on the above findings, if DEFA1, which is specifically expressed and secreted by immune cells, can be used as a biological therapeutic and corresponding specific therapeutic drugs can be developed, it will be of great significance for the treatment and prevention of new coronavirus infection.

[0006] Therefore, the present invention proposes a protein molecule DEFA1 that inhibits novel coronavirus infection. Another object of the present invention is to provide an application of DEFA1 for treating and / or improving diseases related to novel coronavirus infection.

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a protein molecule suitable for the treatment of new coronavirus infection: DEFA1.

[0008] The present invention provides the use of DEFA1 protein and / or its expression activator in the preparation of a drug for treating novel coronavirus infection.

[0009] Furthermore, the use provided by the present invention is characterized in that: the activator includes an agent that activates the expression of DEFA1 protein and / or an agent that promotes the function of DEFA1 protein.

[0010] Furthermore, the application provided by the present invention is characterized in that: the reagent for activating the expression product of DEFA1 protein comprises at least one of the following active components:

[0011] A. Recombinant vector of DEFA1 encoding gene;

[0012] B.DEFA1 recombinant protein;

[0013] C. Inhibition of siRNA targeting DEFA1 protein expression.

[0014] In addition, the present invention also provides a pharmaceutical composition for treating novel coronavirus infection, characterized in that the pharmaceutical composition comprises any of the above-mentioned agents.

[0015] More specifically, the expression and purification of DEFA1 in the present invention is undertaken by a third party company, and the sequence used in the expression plasmid is the CDS region of DEFA1, and the nucleotide sequence is shown in SEQ ID NO 1. The protein amino acid sequence is shown in SEQ ID NO 2.

[0016] SEQ ID NO 1:

[0017] DNA

[0018] CDS Sequence 285

[0020] atgaggaccctcgccatccttgctgccattctcctggtggccctgcaggcccaggctgagccac tccaggcaagagctgatgaggttgctgcagccccggagcagattgcagcggacatcccagaagtggttgtttcccttgcatgggacgaaagcttggctccaaagcatccaggctcaaggaaaaacatggcctgcta ttgcagaataccagcgtgcattgcaggagaacgtcgctatggaacctgcatctaccagggaagactctgggcattctgctgctga

[0021] SEQ ID NO2:

[0022] Amino Acids

[0023] CDS translation sequence 94

[0025] MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKNMACYCRIPACIAGERRYGTCIYQGRLWAFCC

[0026] In addition, the present invention also provides a method for treating and / or improving novel coronavirus infection using DEFA1, comprising two methods:

[0027] A. The DEFA1 expression plasmid was transfected into human peripheral blood mononuclear cells THP-1 to obtain monocytes with high expression of DEFA1.

[0028] B. DEFA1 protein was used to treat SARS-CoV-2-infected human lung adenocarcinoma Calu3 cells or colon adenocarcinoma CaCO2 cells, placing these two types of target cells in an environment with high DEFA1 protein products.

[0029] The transfection steps in step A are as follows: In the clean bench, add 97μl DMEM to a 1ml OD tube, and add 3μg of overexpression DEFA1 plasmid to DMEM, mix gently with a pipette, and let stand at room temperature for 5 minutes. At the same time, add 4μl fugene6 to the premixed DMEM, mix gently and let stand for 15 minutes. After completion, use a 100μl gun to aspirate the reaction solution, evenly add it to a 6-well plate, tap it gently and put it in the incubator. Observe the cell state after 24 hours, discard the cell supernatant, replace it with fresh culture medium, and obtain oe DEFA1-THP-1.

[0030] Intervention treatment in step B: In a clean bench, add at least 100 μg of DEFA1 expression protein to 2 ml of DMEM medium containing 10% FBS (Gibco), 1% L-glutamine and double antibody, and mix thoroughly. Calu3 cells or CaCO2 cells are infected with SARS-CoV-2 virus, and the cell supernatant is discarded after 2 hours, and the culture medium premixed with DEFA1 expression protein is added and placed in an incubator for culture.

[0031] After 24 hours of culture, the SARS-CoV-2 viral RNA level and viral protein expression in Calu3 or CaCO2 cells were detected, and the changes in related inflammatory factors were detected. The experiment was repeated 3 times, and it was found that compared with the control group, the infection of Calu3 or CaCO2 cells with the new coronavirus was significantly inhibited, the viral RNA level in the target cells decreased, the viral protein expression was disturbed, the level of related inflammatory factors was reduced, and the protection effect of the target cells was significantly enhanced after the intervention of DEFA1 expression protein.

[0032] The advantages of the present invention are:

[0033] The present invention can delay the progression of the disease in patients infected with the new coronavirus to the greatest extent and reduce the occurrence of related injuries through interventional treatment. The present invention has discovered for the first time the protein molecule DEFA1 with high therapeutic value for new coronavirus infection; through the development and application of DEFA1-related drugs, the early diagnosis and treatment of new coronavirus infection can be made more convenient and easy, laying the foundation for improving clinical treatment effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 After DIA proteomics research, the proteins of the control group (normal human serum) and the infection group (serum of patients infected with the new coronavirus) were analyzed, and the differentially expressed DEFA1 protein gene was expressed in infected target cells and co-cultured monocytes by real-time PCR verification. *, P<0.05; **, P<0.01. (Figure A: Differential protein expression heat map of serum proteomics DIA sequencing results, Figure B: Verification of the expression of the screened differentially expressed DEFA1 in CaCO2 infected with the new coronavirus and co-cultured THP1 cells, Figure C: Verification of the expression of the DEFA1 gene in Calu3 infected with the new coronavirus and co-cultured THP1 cells)

[0035] Figure 2 In order to simulate the in vivo antiviral environment through the established SARS-CoV-2 target cell and monocyte co-culture system, the correlation between DEFA1 level in monocytes and virus infection was detected by real-time PCR, ELISA and immunofluorescence, and statistical analysis was performed. From the results, we can see that the expression of monocyte DEFA1 is significantly higher than that of the control group as the co-culture time with infected target cells increases, and its antiviral effect also increases, suggesting that DEFA1 may have the value of treating SARS-CoV-2 infection and is worthy of further study. *, P<0.05; **, P<0.01. (Figure A: Expression of viral proteins in Calu3 cells after co-culture of Calu3 and THP1 cells for different time periods, Figure B: Changes in viral RNA levels in Calu3 cells after co-culture of Calu3 and THP1 cells for different time periods, Figure C: Expression of viral proteins in CaCO2 cells after co-culture of CaCO2 and THP1 cells for different time periods, Figure D: Changes in viral RNA levels in CaCO2 cells after co-culture of CaCO2 and THP1 cells for different time periods, Figure E: DEFA1 protein levels in the supernatant of cells after co-culture of Calu3 and THP1 cells for different time periods, Figure F: DEFA1 protein levels in the supernatant of cells after co-culture of CaCO2 and THP1 cells for different time periods)

[0036] Figure 3Different concentrations of exogenous DEFA1 protein were used to treat target cells infected with SARS-CoV-2 virus. The results showed that with the increase of DEFA1 treatment concentration, viral infection in target cells was significantly inhibited, indicating the role of DEFA1 in antiviral. **, P<0.01. (Figure A: Effects of exogenous administration of different concentrations of DEFA1 protein on novel coronavirus infection) DETAILED DESCRIPTION

[0037] The present invention will now be described in detail in conjunction with the embodiments and drawings, but the implementation of the present invention is not limited thereto.

[0038] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to conventional conditions, or according to the conditions recommended by the manufacturer.

[0039] Example 1

[0040] 1. Research subjects

[0041] Peripheral blood of patients infected with the novel coronavirus (n=8) and normal subjects (n=20) were selected to separate serum for high-throughput non-labeled proteomics research and bioinformatics analysis. At the same time, mononuclear cells were separated by blood cell separation technology for transcriptome sequencing and bioinformatics analysis. There was no statistically significant difference in the distribution of factors such as age, smoking, and drinking between the infection group and the control group (P>0.05).

[0042] 2. Research Methods

[0043] 1. Place the peripheral blood of patients or normal people in a 37°C environment to promote coagulation. After the blood coagulates, balance it and centrifuge it (generally at 3000 rpm for 5 to 10 minutes) to obtain serum, which should be carefully aspirated.

[0044] 2. Add 100 μL of lysis buffer (7 M urea, 2 M thiourea) to each sample, followed by sonication to extract total protein.

[0045] 4. Determine protein concentration using the Qubit Fluorescent Protein Quantitation Kit (Invitrogen) according to the manufacturer's instructions.

[0046] 5. Further label-free proteomics studies were performed by Shanghai Zhongke New Life Science Technology Co., Ltd. (Shanghai, China).

[0047] 6. GO analysis was performed on the abnormally expressed proteins, and the top three highly enriched GO groups were screened by Venn analysis from the three aspects of GO analysis: biological process, cellular component and molecular function.

[0048] 7. Peripheral blood of patients or normal people is added to Percoll solution of different concentrations (densities), centrifuged at 400g for 20-25min. Mononuclear cells are recovered from the corresponding layers and washed twice with PBS.

[0049] 8. TRIZol cell lysis buffer was added to each sample to extract total cellular RNA, and transcriptome sequencing and bioinformatics analysis were performed on these samples by Shanghai Liebing Biopharmaceutical Technology Co., Ltd. (Shanghai, China).

[0050] 9. The expression levels of the three selected genes were verified in the target cell and monocyte co-culture system established in vitro. First, the new coronavirus target cells Calu3 or CaCO2 cells were planted in a 24-well plate, a Transwell plate was placed on top, and THP1 cells in suspension culture were placed to establish a co-culture system. The new coronavirus was added to the supernatant of the target cells, the medium was changed after 6 hours of infection, and the culture was continued for 12, 24 or 36 hours. After centrifugation of the suspended THP1 cells and removal of the supernatant from the target cells, TRIZol was added to lyse and extract total cell RNA. Subsequently, RT-PCR was used to detect the expression level of DEFA1 in each cell.

[0051] Real-time RT-PCR (qPCR) method

[0052] (1) The primer sequences are as follows;

[0053] DEFA1:

[0054] Forward primer: 5'TGCTTTGAGAGCTACAGGGA3' (SEQ ID NO 3)

[0055] Reverse primer: 5'TGGTACGTGTATCCCAAGTGTC3' (SEQ ID NO 4)

[0056] GAPDH:

[0057] Forward primer: 5'AGTTGCGTTACACCCTTTCTTG 3' (SEQ ID NO 5)

[0058] Reverse primer: 5'GCTGTCACCTTCACCGTTCC 3' (SEQ ID NO 6)

[0059] (2) According to TB Premix Ex TaqTM The kit instructions were used for real-time fluorescence quantitative PCR detection and analysis. The qPCR system in each well was 20 μl, including: 10 μl 2×SYBR Master Mix, 0.4 μl of upstream and downstream primers (10 μM) and 8.8 μl of deionized water. Try to avoid and reduce the generation of bubbles in the system, mix thoroughly and centrifuge for 1 min (700 rpm). Amplify according to the following amplification conditions:

[0060]

[0061] Each experiment was repeated at least three times independently.

[0062] (3) Results The relative mRNA content of each gene was compared by calculating the 2-ΔΔCT value based on the ΔCT value, and the t-test was used. P < 0.05 was considered statistically significant, and P < 0.01 was considered a significant difference.

[0063] 3. Experimental Results

[0064] The differentially expressed proteins in the serum of patients with COVID-19 were screened by serum non-labeled proteomics sequencing. Finally, the DEFA1 protein with obvious abnormal expression was screened. The mRNA expression of DEFA1 in the infected and non-infected states was detected at the target cell and monocyte levels in the in vitro co-culture system. As shown in Figures 1B and 1C, the expression of DEFA1 in monocytes co-cultured with infected target cells was significantly increased compared with the control.

[0065] Example 2: The expression of DEFA1 molecules is correlated with novel coronavirus infection

[0066] 1. Research subjects

[0067] Correlation between viral RNA and protein expression levels in SARS-CoV-2 target cells Calu3 or CaCO2 and DEFA1 levels in co-cultured THP-1 monocytes and supernatants.

[0068] 2. Research Methods

[0069] 1. Establish a co-culture system of SARS-CoV-2 target cells Calu3 or CaCO2 and monocytes to simulate the situation of immune cells gathering around infected tissues after infection in vivo. The method is to connect the SARS-CoV-2 target cells Calu3 or CaCO2 cells and monocytes THP1 through the upper and lower chambers of Transwell to establish a co-culture system. Add the SARS-CoV-2 to the supernatant of the target cells for infection for 6 hours, and continue to culture for 12, 24, or 36 hours.

[0070] 2. Detect the expression level of the new coronavirus nucleocapsid protein in the target cells at different time points after co-culture to reflect the status of viral infection. Specific method of immunofluorescence detection: remove the supernatant of the target cells, add methanol to each well, and incubate at 4°C for 30 minutes to fix the cells. Remove the methanol, wash with PBS three times, and add 3% BSA for blocking. Subsequently, add the new coronavirus nucleocapsid protein monoclonal antibody (ThermoFisher) and incubate at room temperature for 2 hours. After removing the antibody, wash with PBS three times and add rabbit anti-mouse Alexa The cells were incubated with 488 secondary antibody (Abcam) at room temperature for 1 hour. Immunofluorescence detection was performed after nuclear staining with DAPI.

[0071] 3. Detect the level of SARS-CoV-2 RNA in target cells at different time points after co-culture to reflect the virus infection. The specific RT-qPCR detection method is the same as described in Example 1.

[0072] The primer sequences are as follows;

[0073] SARS-CoV-2:

[0074] Forward primer: 5'GGGGAACTTCTCCTGCTAGAAT 3' (SEQ ID NO 7)

[0075] Reverse primer: 5'CAGACATTTTGCTCTCAAGCTG 3' (SEQ ID NO 8)

[0076] 4. ELISA (Reddot Biotech) was used to detect the expression of DEFA1 in the co-culture supernatant and analyze the correlation between the two. Specific method of ELISA detection: Add 0.1 ml of the sample to be tested to the coated reaction well and incubate at 37°C for 1 hour. Then wash. (Blank wells, negative control wells and positive control wells are also made at the same time). Then add 0.1 ml of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and then wash. Add 0.1 ml of the prepared TMB substrate solution to each reaction well and incubate at 37°C for 30 minutes. Then, add 0.05 ml of 2M sulfuric acid to each reaction well to terminate the reaction. On the microplate reader, at 450 nm, measure the OD value of each well after adjusting the blank control well to zero, and calculate the content of DEFA1 in each well using the standard curve formula.

[0077] 3. Research Results

[0078] In the co-culture system, it can be observed that as the co-culture time of infected target cells and monocytes increases, the expression of viral proteins in target cells decreases significantly, and the level of viral RNA is significantly suppressed. At the same time, the level of DEFA1 in the co-culture supernatant increases significantly with the increase of co-culture time. Figure 2The results show that the expression and secretion of DEFA1 in monocytes is correlated with the anti-new coronavirus infection effect.

[0079] Example 3: DEFA1 molecules have the effect of inhibiting the infection of new coronavirus

[0080] 1. Research subjects

[0081] Same as Example 2.

[0082] 2. Research Methods

[0083] 1. After Calu3 or CaCO2 cells were infected with SARS-CoV-2 virus, different concentrations (0.08, 0.4, 2, 10, 50 μg / ml) of exogenous DEFA1 protein were added, and the effect on virus-infected target cells was detected by immunofluorescence method 36 hours after infection. The specific method of immunofluorescence detection is the same as described in Example 2.

[0084] 3. Research Results

[0085] Specific as Figure 3 As shown. As the concentration of exogenously administered DEFA1 increases, the anti-new coronavirus effect in target cells also significantly increases. The significant antiviral activity of DEFA1 is confirmed.

[0086] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

[0087]

[0088]

[0089]

[0090]

[0091] Sequence Listing <110> Chinese People's Liberation Army Naval Medical University <120> Application of DEFA1 in COVID-19 Infection <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 285 <212> DNA <213> CDS sequence (Artificial) <400> 1 atgaggaccc tcgccatcct tgctgccatt ctcctggtgg ccctgcaggc ccaggctgag 60 ccactccagg caagagctga tgaggttgct gcagccccgg agcagattgc agcggacatc 120 ccagaagtgg ttgtttccct tgcatgggac gaaagcttgg ctccaaagca tccaggctca 180 aggaaaaaca tggcctgcta ttgcagaata ccagcgtgca ttgcaggaga acgtcgctat 240 ggaacctgca tctaccaggg aagactctgg gcattctgct gctga 285 <210> 2 <211> 94 <212> PRT <213> CDS translated sequence (Artificial) <400> 2 Met Arg Thr Leu Ala Ile Leu Ala Ala Ile Leu Leu Val Ala Leu Gln 1 5 10 15 Ala Gln Ala Glu Pro Leu Gln Ala Arg Ala Asp Glu Val Ala Ala Ala 20 25 30 Pro Glu Gln Ile Ala Ala Asp Ile Pro Glu Val Val Val Ser Leu Ala 35 40 45 Trp Asp Glu Ser Leu Ala Pro Lys His Pro Gly Ser Arg Lys Asn Met 50 55 60 Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr 65 70 75 80 Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys 85 90 <210> 3 <211> 20 <212> DNA <213> Artificial <400> 3 tgctttgaga gctacaggga 20 <210> 4 <211> 22 <212> DNA <213> Artificial <400> 4 tggtacgtgt atcccaagtg tc 22 <210> 5 <211> 22 <212> DNA <213> Artificial <400> 5 agttgcgtta caccctttct tg 22 <210> 6 <211> 20 <212> DNA <213> Artificial <400> 6 gctgtcacct tcaccgttcc 20 <210> 7 <211> 22 <212> DNA <213> Artificial <400> 7 ggggaacttc tcctgctaga at 22 <210> 8 <211> 22 <212> DNA <213> Artificial <400> 8 cagacatttt gctctcaagc tg 22

Claims

1. Application of DEFA1 protein in the preparation of drugs for the treatment of novel coronavirus infection.

Images