A monoclonal antibody for NS1 protein and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody for NS1 protein and its application. The amino acid sequence of the heavy chain variable region of the monoclonal antibody for NS1 protein is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2. The present invention provides a specific monoclonal antibody for the non-structural protein NS1 protein of respiratory syncytial virus, and the monoclonal antibody is an IgG1 type monoclonal antibody; Western blotting test shows that it specifically reacts with the non-structural protein NS1; indirect immunofluorescence test shows that it has good reactivity with RSV A2 strain.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a NS1 protein monoclonal antibody and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is a seasonal virus that is a common cause of lower respiratory tract infections in infants, the elderly, and those with underlying medical conditions that increase the risk of RSV. According to statistics, most children will experience at least one RSV infection by the age of 2 years.

[0003] RSV is a non-segmented single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae, genus Pneumovirus, subfamily Pneumovirinae, with a diameter of about 150 nm, a genome of 15.2 kb, 10 genes, encoding 11 open reading frames (ORFs), because there are two overlapping open reading frames in M2 mRNA, which produce two different matrix proteins, M2-1 and M2-2. The viral envelope contains four proteins, matrix protein (M), G glycoprotein, fusion (F) glycoprotein and small hydrophobic protein (SH). RSV virus includes five ribosomal proteins, polymerase subunit protein (L), nucleocapsid (N), phosphoprotein (P), M2-1 and M2-2, and two non-structural proteins (NS1 and NS2).

[0004] The NS1 gene is 417 bp in length, encoding 139 amino acid residues and a protein size of 15.6 kDa. NS1 is believed to have multiple roles in RSV pathogenesis. In RSV-infected A549 cells, NS1 inhibits the expression of miR-24 by upregulating the transcription factor Kruppel-like factor-6, which drives the expression of TGF-β. Notably, inhibition of miR-24 can actually inhibit RSV replication in A549 cells. It is possible that NS1 inhibits RSV-induced upregulation of miR-24 within 24 hours after infection, and subsequently, miR-24 expression is upregulated to enhance viral replication. In addition to infecting airway epithelial cells, RSV can also infect dendritic cells and activate PRRs through PAMPs. Infection with wild-type and NS-deficient RSV strains showed that NS1 inhibited the maturation of human dendritic cells. In addition, MAVS, an essential signaling protein for RSV-induced type I IFN production, was also strongly inhibited. Other studies have shown that the loss of NS1 can promote CD103 + CD8 + Proliferation and activation of T cells and Th17 cells, two cell populations that can counteract RSV and inhibit IL-4-producing CD4 + Activation of T cells.

[0005] The crystal structure of NS1 was determined in 2017, and it has significant structural homology with the N-terminal domain of the RSV matrix protein (M). An obvious difference between NS1 and M is the presence of an α-helix (α3) at the C-terminus of NS1. Studies have shown that mutations in the C-terminal α-helix do not affect the nuclear localization of NS1, but the interaction of the α3 helix with other proteins is important. The NS1 part lacking the α3 helix eliminates the NS1-mediated IFN-β-induced inhibition and also loses its antagonistic function on dendritic cell maturation, indicating that this unique helical structure is essential for NS1-mediated IFN antagonism and regulation of immune responses. Compared with wild type (WT) RSV infection, the recombinant strain with a mutated NS1 C-terminal α-helix has a reduced viral replication rate in A549 cells. Current research results confirm that NS1 is distributed in both the cytoplasm and the nucleus. After entering the nucleus, NS1 interacts with mediators, binds to the promoters and enhancers of host immune genes, regulates the transcription of host immune genes, and thus antagonizes the host's antiviral immune response. However, the exact molecular mechanism of this inhibition has not yet been clarified. Therefore, further research on the interaction between non-structural protein NS1 and host factors will help to systematically understand the molecular mechanisms of RSV infection and its immune escape.

[0006] Since there is no commercial anti-RSV NS1 monoclonal antibody (mAb), in-depth research on the function and regulatory mechanism of NS1 protein is limited. In this study, NS1 mAb was successfully prepared and used for indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressing cells and virus-infected cells, providing a useful tool for further studying the interaction mechanism between non-structural protein NS1 and host proteins during RSV infection. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a NS1 protein monoclonal antibody and application thereof. The NS1 monoclonal antibody provided by the present invention can specifically bind to RSV NS1 protein.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention also provides a NS1 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the NS1 protein monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0010] Preferably, the nucleotide sequence of the heavy chain variable region of the NS1 protein monoclonal antibody is as shown in SEQ ID No.3.

[0011] Preferably, the amino acid sequence of the light chain variable region of the NS1 protein monoclonal antibody is as shown in SEQ ID No.4.

[0012] Preferably, the CDR sequence of the heavy chain variable region of the NS1 protein monoclonal antibody is shown as SEQ ID No. 5-7.

[0013] Preferably, the CDR sequence of the light chain variable region of the NS1 protein monoclonal antibody includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence;

[0014] The light chain CDR1 sequence is shown in SEQ ID No.8;

[0015] The light chain CDR2 sequence is KVS;

[0016] The light chain CDR3 sequence is shown as SEQ ID No.9.

[0017] Preferably, the NS1 protein monoclonal antibody is obtained by immunizing animals with RSV NS1 protein.

[0018] Preferably, the amino acid sequence of the RSV NS1 protein is shown in SEQ ID No.10.

[0019] The present invention also provides the use of the NS1 protein monoclonal antibody described in the above technical solution in the preparation of a drug that specifically binds to RSV NS1 protein.

[0020] The present invention also provides a drug for treating respiratory syncytial virus, comprising the NS1 monoclonal antibody described in the above technical solution.

[0021] Preferably, it also includes a pharmaceutically acceptable carrier.

[0022] Beneficial effects of the present invention:

[0023] The present invention provides a specific monoclonal antibody against the non-structural protein NS1 of respiratory syncytial virus, wherein the monoclonal antibody is an IgG1 monoclonal antibody;

[0024] Western blotting experiments showed that it specifically reacted with the nonstructural protein NS1;

[0025] Indirect immunofluorescence test showed that it had good reactivity with RSV A2 strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0027] Figure 1 Detection of RSV NS1 protein by SDS-PAGE; M: protein molecular weight standard; 1: precipitate of induced protein after ultrasound; 2: supernatant of induced protein after ultrasound; 3-4: washing solution; 5-6: eluent; 7: NS1 protein with tag removed;

[0028] Figure 2 Purification of NS1 antibody for SDS-PAGE detection; M: protein molecular weight standard; 1: purified NS1 antibody;

[0029] Figure 3 Purification of NS1 antibody for Western blotting identification; M: protein molecular weight standard; 1: purified RSV NS1 protein; 2: purified RSV NS2 protein;

[0030] Figure 4 Western blotting identification of eukaryotic protein NS1; M: protein molecular weight standard; 1: lysate of 293T cells transfected with pCAGGS-HA empty plasmid; 2: lysate of 293T cells transfected with pCAGGS-HA-NS1 plasmid; 3: lysate of 293T cells transfected with pCAGGS-HA-NS2 plasmid;

[0031] Figure 5 Western blotting identification of NS1 monoclonal antibody for RSV infection; M: protein molecular weight standard; 1: A549 cell lysate infected with RSV; 2: A549 cell lysate not infected with RSV;

[0032] Figure 6 The expression and subcellular localization of NS1 protein in transfected cells;

[0033] Figure 7 The expression and subcellular localization of RSV NS1 protein in virus-infected cells. DETAILED DESCRIPTION

[0034] The present invention provides a NS1 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the NS1 protein monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0035] SEQ ID No.1:

[0036] EVQLQESGGGLVQPGGSMKLSCVASGFTFSNYWLNWVRQSPEKGLEWVATIRLKSTNYATDYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRSGYYPLDHWGQGTTLTVSS.

[0037] In the present invention, the nucleotide sequence of the heavy chain variable region of the NS1 protein monoclonal antibody is shown as SEQ ID No.3.

[0038] SEQ ID No.3:

[0039] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGTTGAACTGGGTCCCGCCAGTCTCCAGAGAAGGGACTTGAATGGGTTGCTACAATTAGATTGAAATCTACTAATTATGCAAC AGATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAGATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGAGTGGTTACTACCCCCTTGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG.

[0040] SEQ ID No.2:

[0041] DIVLTQTPLSLPVSLGDQASISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYKVSDRLSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK.

[0042] In the present invention, the amino acid sequence of the light chain variable region of the NS1 protein monoclonal antibody is shown in SEQ ID No.4.

[0043] SEQ ID No.4:

[0044] GATATTGTGCTCACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTATATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT CCGACCGATTATCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC.

[0045] In the present invention, the CDR sequences of the heavy chain variable regions of the NS1 protein monoclonal antibodies are shown in SEQ ID Nos. 5-7.

[0046] SEQ ID No.5:

[0047] GFTFSNYW;

[0048] SEQ ID No.6:

[0049] IRLKSTNYAT;

[0050] SEQ ID No.7:

[0051] TRSGYYPLDH.

[0052] In the present invention, the CDR sequence of the light chain variable region of the NS1 protein monoclonal antibody includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence;

[0053] The light chain CDR1 sequence is shown in SEQ ID No. 8, QSIVYSNGNTY;

[0054] The light chain CDR2 sequence is KVS;

[0055] The light chain CDR3 sequence is shown in SEQ ID No.9, FQGSHVPWT.

[0056] In the present invention, the NS1 protein monoclonal antibody is obtained by immunizing an animal with RSV NS1 protein. The present invention does not specifically limit the method for obtaining the NS1 protein monoclonal antibody by immunizing an animal with RSV NS1 protein, and those skilled in the art can use conventional methods.

[0057] In the present invention, the amino acid sequence of the RSV NS1 protein is shown in SEQ ID No. 10, which is as follows:

[0058] SEQ ID No.10:

[0059] MGSNSLSMIKVRLQNLFDNDEVALLKITCYTDKLIHLTNALAKAVIHTIKLNGIVFVHVITSSDICPNNNNIVVKSNFTTMPVLQNGGYIWEMMELTHCSQPNGLIDDNCEIKFSKKLSDSTMTNYMNQLSELLGFDLNP.

[0060] The present invention also provides the use of the NS1 protein monoclonal antibody described in the above technical solution in the preparation of a drug that specifically binds to RSV NS1 protein.

[0061] The present invention also provides a drug for treating respiratory syncytial virus, comprising the NS1 monoclonal antibody described in the above technical solution. In the present invention, the drug preferably also comprises a pharmaceutically acceptable carrier.

[0062] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0063] The genes, strains, plasmids, viruses and experimental animals used in the following examples are as follows: the sequence of RSV NS1 gene was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., Escherichia coli (E. coli) DH5α and BL21 (DE3) competent cells were purchased from TaKaRa, plasmids pCAGGS-HA, pCAGGS-Flag, pCAGGS-HA-NS1, pCAGGS-flag-NS2, 293T cells, A549 cells and RSV A2 strain were preserved in this laboratory, and 6-8 week old female BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0064] The main reagents used in the following examples are: restriction endonucleases Bam HI and XII was purchased from TaKaRa; HA-tagged mouse monoclonal antibody, Flag-tagged mouse monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, Freund's incomplete adjuvant, Freund's complete adjuvant, fetal bovine serum, Flag beads, and Flag peptide were all purchased from Sigma; HisTrap FF nickel affinity chromatography column and protein G purification column were purchased from Cytiva; small ubiquitin-related modifier (SUMO) protease was purchased from Solebao Technology Co., Ltd. (Beijing); and ECL color development kit was purchased from Merck.

[0065] Example 1

[0066] With reference to the RSV gene published by NCBI (mutant human respiratory syncytial virus strain long, accession number: KF713490.1), the codons of the NS1 full gene were optimized and restriction sites were introduced at both ends of the expression sequence. Bam HI and XI I, the gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and constructed into the prokaryotic expression vector pET-28a-HisSUMO, and the constructed recombinant plasmid was named pET-28a-HisSUMO-NS1, which was transformed into E. coli DH5α competent cells, after plasmid extraction Bam HI and XI The DNA was identified by double enzyme digestion with I and sequenced and verified by Beijing Qingke Biotechnology Co., Ltd. (Xi'an).

[0067] The recombinant plasmid pET-28a-HisSUMO-NS1 was transformed into E. coliBL21(DE3) competent cells were selected and the positive monoclonal colonies were inoculated into 5 mL of LB liquid medium containing kanamycin (100 μg / mL) and cultured overnight at 37°C and 220 rpm. The glycerol bacteria were stored and the bacterial liquid was inoculated into 100 mL of LB liquid medium containing kanamycin at a ratio of 1:50. When the OD600 was about 0.6, 100 μL of 1 M IPTG was added and the expression was induced at 16°C for 14 h. The bacteria were collected by centrifugation at 8 000 r / min for 10 min, and the bacterial precipitate was resuspended with a balance buffer solution (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) and ultrasonically disrupted. The supernatant and precipitate were collected separately, and the precipitate was resuspended and dissolved with 8 M urea. After the samples were prepared, the expression of NS1 protein was observed by SDS-PAGE electrophoresis.

[0068] The stored glycerol bacteria were induced to express in large quantities according to the above steps, and the cells were collected by centrifugation. After ultrasonic disruption, the cell supernatant was collected by centrifugation. The nickel column was connected to the AKTA pure 150 purifier, and the collected supernatant was filtered through a 0.45 μm filter membrane and purified by affinity chromatography. The impurities were washed and eluted with a linear gradient of imidazole buffer, and the washing liquid and the elution liquid were collected respectively. The purified fusion protein was digested with SUMO protease at room temperature for 3 h, and further purified by affinity chromatography to remove the His-SUMO protein tag. The protein purification was detected by SDS-PAGE.

[0069] According to the affinity chromatography purification method, the RSV NS1 protein was successfully purified by SDS-PAGE analysis. The size of the purified protein was consistent with the expected protein molecular weight. The results are shown in Figure 1 , 139 amino acid residues, the protein size is 15.6 kDa, the isoelectric point is 5.92, and its amino acid sequence is shown in SEQ ID No.10.

[0070] SEQ ID No.10:

[0071] MGSNSLSMIKVRLQNLFDNDEVALLKITCYTDKLIHLTNALAKAVIHTIKLNGIVFVHVITSSDICPNNNNIVVKSNFTTMPVLQNGGYIWEMMELTHCSQPNGLIDDNCEIKFSKKLSDSTMTNYMNQLSELLGFDLNP.

[0072] Example 2

[0073] To prepare monoclonal antibodies against RSV NS1 protein, the purified NS1 protein was emulsified with adjuvant at a ratio of 1:1 to prepare a vaccine. Six female BALB / c mice aged 6-8 weeks were immunized with Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the second to fourth immunizations. Immunizations were performed on days 0, 14, 28, and 36, respectively, with a protein dose of 50 μg per mouse each time. 14 days after the fourth immunization, the mice were tail-cut and blood was collected. The serum was separated and the antibody titer was determined.

[0074] RSV NS1 protein was used as the coating antigen, and the antigen was diluted with coating buffer and added to the ELISA microplate at 100 ng / well and 100 μL / well. The plate was incubated at 4°C overnight. The ELISA plate coated with the antigen was removed, the solution was discarded, the plate was washed 5 times with 1×PBST, and patted dry. 2% bovine serum albumin (BSA) was added at 200 μL / well, blocked at 37°C for 2 h, washed 5 times with 1×PBST, and patted dry. SP2 / 0 mouse ascites prepared was used as a negative control, and mouse serum (2-fold gradient dilution starting from 1:1 000) was used as the primary antibody, which was diluted 2-fold with 1×PBST (starting from 1:1 000) and added to the ELISA plate at 100 μL / well. Incubated at 37°C for 1 h, the solution was discarded, the plate was washed 5 times with 1×PBST, and patted dry. Dilute HRP-labeled goat anti-mouse IgG with 1×PBST (1:10 000) and add to each well, 100 μL / well, incubate at 37℃ for 1 h, discard the solution, wash the plate 5 times and pat dry. Add TMB substrate colorimetric solution, 50 μL / well, incubate at 37℃ in the dark for 15 min. Add stop solution, 50 μL / well, and measure the OD450 value on the microplate reader within 5 min. When (OD value of the experimental sample well sample - OD value of the blank control) / (OD value of the negative control - OD value of the blank control) ≥ 2.1, that is, when S / N ≥ 2.1, it is judged as positive.

[0075] The mouse spleen cells with the highest antibody level were used for fusion test with SP2 / 0 cells. After fusion, the indirect ELISA method was used, with the hybridoma cell supernatant as the primary antibody and HRP-labeled goat anti-mouse IgG antibody as the secondary antibody to screen the positive hybridoma cell lines that reacted with RSV NS1. The positive hybridoma cell lines were cloned and purified three times and then cultured for subculture. The cells were collected and 2×10 6 Cells / branch were stored in liquid nitrogen.

[0076] One week before the injection of monoclonal cell lines, sterile liquid paraffin was injected intraperitoneally, and each BALB / c mouse was injected with 500 μL of liquid paraffin. Hybridoma monoclonal cells with good immunoreactivity were collected, and after cell counting, the cell density was adjusted to 1×10 6 / ml. Subsequently, the cell suspension was injected into the peritoneal cavity of BALB / c mice, and the ascites was collected multiple times within 10 to 14 days. The mouse ascites was centrifuged at 12000 rpm for 10 minutes, and impurities such as lipids and tissue fragments were removed by filtration to obtain clear ascites. The antibody was purified by affinity chromatography. The purification method is as follows:

[0077] (1) Column pretreatment: Rinse the Protein G affinity column 3-5 times with 10 column volumes of deionized water at a flow rate of 1 ml / min.

[0078] (2) Equilibration: Rinse the column 3-5 times with 10 column volumes of equilibration buffer (0.02M PB + 0.3M NaCl, pH 7.0) at a flow rate of 1 ml / min until the pH value of the effluent remains unchanged.

[0079] (3) Sample loading: Dilute the ascites with 0.02 M PB, filter through a 0.22 μm filter membrane, and set the flow rate to 0.6 mL / min for sample loading.

[0080] (4) Washing: After loading, wash the column with 5-10 times the column volume of equilibration buffer and collect the effluent.

[0081] (5) Elution: Elute with 5-10 times elution buffer (0.1 M glycine, pH 3.0) and collect the effluent.

[0082] (6) pH adjustment: After elution, immediately adjust the pH of the eluted product to neutral with saturated sodium carbonate.

[0083] (7) Cleaning the chromatography column: Rinse the chromatography column with 5-10 times the column volume of pure water, and then immediately rinse with 5 times the equilibration buffer to equilibrate the chromatography column to neutrality.

[0084] (8) Storage of chromatography columns: After flushing the affinity chromatography column with 5 column volumes of 20% ethanol, store at 4°C.

[0085] (9) Sample concentration: Use a 10 kDa ultrafiltration tube to concentrate the eluted product to about 1-5 mL.

[0086] (10) Dialysis: Add all concentrated samples into a dialysis bag and place them in 5 L buffer (0.01 M PBS, pH = 7.4) for overnight dialysis. Change the solution once on the second day.

[0087] (12) Antibody purity was analyzed by SDS-PAGE and protein content was determined.

[0088] The results of SDS-PAGE analysis are shown in Figure 2The antibody heavy chain and light chain bands were shown at 50 kDa and 25 kDa, respectively. The content of the purified NS1 monoclonal antibody was 7.5 mg / mL.

[0089] (13) Western blotting results are shown in Figure 3 , indicating that the screened NS1 monoclonal antibody has good reactivity with the purified NS1 protein.

[0090] (1) The monoclonal antibody Ig subtype was identified using a mouse monoclonal antibody subtype identification kit (Proteintech), and the IgG1 subtype of the NS1 monoclonal antibody was measured.

[0091] (2) Recovery of hybridoma cells: Take out a hybridoma cell cryopreservation tube containing a specific monoclonal antibody against the respiratory syncytial virus NS1 protein from the liquid nitrogen tank, clamp it with tweezers and quickly place it in a 37°C water bath. Shake it continuously to thaw the cells quickly. Open the cryopreservation tube aseptically and transfer the cell fluid into 10 mL of RPMI-1640 culture medium. Place it in a 37°C incubator containing 5% CO2 until the hybridoma cells are completely attached to the wall, and then change the cell medium.

[0092] (3) PCR amplification of the variable region of the monoclonal antibody: The cells were collected and centrifuged at 1500 rpm for 10 min, and RNA was extracted using the Trizol method. Subsequently, the RNA was reverse transcribed into cDNA using 2× Prime Script RT Max Master Mix. The prepared cDNA was used as a template and its gene fragment was amplified using a high-fidelity polymerase. The system was 25 μL of 2× Rapid Taq Master Mix, 1 μL each of Forward and Reverse, 1 μL of cDNA, and ddH2O was added to 50 μL. The amplification conditions were pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 50 s, 35 cycles; final extension at 72°C for 10 min. After the PCR reaction was completed, the samples were electrophoresed on a 2% agarose gel to detect the results.

[0093] (4) Determination and analysis of antibody variable regions: The heavy chain gene and light chain gene were cloned into the pMD18-T vector, respectively, and a single white colony was selected for culture. The plasmid identified as positive by PCR was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were compared with the antibody gene library (IMGT / v-quest). The complementarity determining region (CDR) sequences of the heavy chain variable region and the light chain variable region of the anti-RSV NS1 protein monoclonal antibody prepared in this example are shown in Table 1.

[0094] Table 1 Antibody variable region sequences

[0095] CDR1 CDR2 CDR3 Heavy chain variable region GFTFSNYW IRLKSTNYAT TRSGYYPLDH Light chain variable region QSIVYSNGNTY KVS FQGSHVPWT

[0096] Among them, the amino acid sequence of the heavy chain variable region is (SEQ ID No. 1):

[0097] EVQLQESGGGLVQPGGSMKLSCVASGFTFSNYWLNWVRQSPEKGLEWVATIRLKSTNYATDYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTRSGYYPLDHWGQGTTLTVSS.

[0098] The gene sequence encoding the heavy chain variable region is (SEQ ID No. 3):

[0099] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGTTGAACTGGGTCCCGCCAGTCTCCAGAGAAGGGACTTGAATGGGTTGCTACAATTAGATTGAAATCTACTAATTATGCAAC AGATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAGATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCAGGAGTGGTTACTACCCCCTTGACCACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG.

[0100] The amino acid sequence of the light chain variable region is (SEQ ID No. 2):

[0101] DIVLTQTPLSLPVSLGDQASISCRSSQSIVYSNGNTYLEWYLQKPGQSPKLLIYKVSDRLSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK.

[0102] The gene sequence encoding the light chain variable region is (SEQ ID No. 4):

[0103] GATATTGTGCTCACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTATATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT CCGACCGATTATCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGTTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC.

[0104] Example 3

[0105] The eukaryotic expression plasmid pCAGGS-HA-NS1 and the empty plasmid pCAGGS-HA were transfected into 293T cells, respectively. The cell samples were collected and processed, and their reactivity with NS1 monoclonal antibody was detected by Western blotting. The specific experimental process is as follows:

[0106] (1) The eukaryotic expression plasmid pCAGGS-HA-NS1 and the empty plasmid pCAGGS-HA were transfected into 293T cells respectively. The 293T cells were collected after culture at 37°C for 24 h.

[0107] (2) Sample preparation: Lyse cells using lysis buffer, centrifuge at 12,000 rpm for 15 min to obtain protein samples, add 5× protein loading buffer, mix thoroughly, place in a 100°C metal bath for 10 min, cool, and centrifuge at 12,000 rpm for 2 min.

[0108] (3) Electrophoresis: Fix the SDS-PAGE gel (12% separation gel, 5% concentration gel) on the electrophoresis device, add 1× Tris-glycine electrophoresis buffer, and load 10 µL per lane. The initial electrophoresis voltage is 80 V. After the sample enters the separation gel, adjust the voltage to 120 V and continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel, then terminate the electrophoresis.

[0109] (4) Transfer: Pre-cool the membrane transfer buffer in a 4°C refrigerator. After removing the concentrated gel, wash the surface with deionized water. Cut a PVDF membrane of the same size as the gel and soak it in 100% methanol for 1-2 min. Stack them neatly in the order of "black surface - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - white surface", paying attention to removing the bubbles between the layers, place them in a wet transfer tank, add enough transfer buffer, and transfer the membrane at a constant voltage of 100 V for 2 hours.

[0110] (5) Blocking: After transfer, remove the PVDF membrane and block it with 5% skimmed milk powder at room temperature for 1-2 h.

[0111] (6) Incubation with primary antibody: Add 5 mL of anti-NS1 mouse monoclonal antibody diluted in 5% skim milk powder (1:4,000) and incubate overnight at 4°C.

[0112] (7) Incubation with secondary antibody: Wash five times with 1× PBST, shaking for 5 min each time at room temperature. Add 5 mL of horseradish peroxidase-labeled goat anti-mouse IgG antibody diluted in 5% skim milk powder (1:5000) and incubate at room temperature for 1 h.

[0113] (8) Development: Wash with 1× PBST five times, shaking for 5 min each time under room temperature conditions, mix ECL substrate colorimetric reagent A and reagent B in a 1:1 ratio, add an appropriate amount of colorimetric solution to the PVDF membrane, scan and expose, and save the image.

[0114] Test results see Figure 4 The NS1 monoclonal antibody of the present invention specifically reacts with the RSV non-structural protein NS1, but not with the RSV non-structural protein NS2 protein, and the 293T cells transfected with the empty vector do not have this band at the same position, indicating that the monoclonal antibody can specifically bind to the NS1 protein. This indicates that the monoclonal antibody NS1 of the present invention can specifically react with the RSV non-structural protein NS1 protein.

[0115] A549 cells were infected with RSV MOI=1 virus, and A549 cells were collected after culturing at 37℃ for 72h, and uninfected cells were used as negative control; cells were lysed with NP-40 lysis buffer, centrifuged at 12000 rpm for 15 min to obtain protein samples, and then protein loading buffer was added, and metal bath was placed at 100℃ for 10 min, and SDS-PAGE gel electrophoresis was performed with 12% protein gel; transfer was performed at 100 V constant voltage for 2h. After the transfer was completed, the PVDF membrane was removed and blocked with 5% skim milk powder at room temperature for 1h; NS1 monoclonal antibody diluted with blocking solution was added, and incubated at 4℃ overnight; washed with 1×PBST 5 times, diluted HRP-labeled goat anti-mouse IgG antibody (1:5000 dilution) was added, and incubated at room temperature for 1h. Washed with 1×PBST 5 times, an appropriate amount of ECL colorimetric solution was added to the PVDF membrane, and the image was saved after scanning and exposure.

[0116] Western blotting results are shown in Figure 5 NS1 monoclonal antibody detected a clear specific reaction band for the protein sample derived from infected A549 cells, while there was no such band at the same position in uninfected A549 cells.

[0117] Example 4

[0118] A549 cells were cultured at 1×10 5 The cells were inoculated at a density of 1:1 / well in a confocal dish, and the eukaryotic expression plasmid pCAGGS-HA-NS1 and the empty plasmid pCAGGS-HA were used to transfect the cells for 24 h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with 0.2% TritonX-100 for 10 min at room temperature, and blocked with 2% BSA blocking buffer at room temperature for 1 h; incubated with RSV NS1 monoclonal antibody (1:500 dilution) at 4 °C overnight; then incubated with goat anti-mouse 488 green fluorescent secondary antibody (1:500 dilution) at 37 °C for 1 h; washed with PBS 4 times, and finally stained with DAPI for 5 min at room temperature, washed with PBS 4 times, and observed and photographed under a confocal microscope. Confocal results ( Figure 6 ) showed that the monoclonal antibody bound well to the RSV NS1 overexpression product in A549 cells with strong specificity, while the empty vector pCAGGS-HA transfection control group showed no fluorescence. In cells transfected with pCAGGS-HA-NS1, the NS1 protein was mainly distributed in the cytoplasm, with a small amount localized in the nucleus.

[0119] A549 cells were adsorbed with RSV MOI=1 virus for 2h, and uninfected cells were used as negative controls. The confocal dish was gently shaken every 30min. After adsorption for 2h, the cells were cultured with complete medium containing 10% fetal bovine serum for 48h. The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30min, washed with PBS for 4 times, and permeabilized with 0.2% TritonX-100 in PBS solution at room temperature for 10min; the solution was discarded, and the cells were blocked with Blocking buffer solution containing 2% BSA at room temperature for 1h; RSV NS2 monoclonal antibody was diluted with the above Blocking buffer solution, and the primary antibody was incubated at 4℃ overnight; PBS was washed 4 times, goat anti-mouse 488 green fluorescent secondary antibody was diluted with PBS, and incubated in a constant temperature incubator at 37℃ for 1h in the dark; PBS was washed 4 times, and the nuclei were stained with DAPI at room temperature in the dark for 5min, and PBS was washed 4 times, and the cells were observed and photographed under a confocal microscope. Results ( Figure 7 ) showed that the NS1 monoclonal antibody had a good specific reaction with the protein samples from RSV-infected A2549 cells, and had no non-specific binding in uninfected A549 cells. The study found that the NS1 protein was mainly distributed in the cytoplasm in RSV-infected cells, with a small amount localized in the nucleus.

[0120] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A monoclonal antibody against RSV NS1 protein, characterized in that: The amino acid sequence of the heavy chain variable region of the RSV NS1 protein monoclonal antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. Use of the RSV NS1 protein monoclonal antibody according to claim 1 in the preparation of a drug for treating respiratory syncytial virus infection.

3. A drug for treating respiratory syncytial virus infection, characterized in that: Comprising the RSV NS1 monoclonal antibody of claim 1.

4. The drug according to claim 3, characterized in that Also included are pharmaceutically acceptable carriers.

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