A monoclonal antibody for NS2 protein and its application

Abstract

The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody to NS2 protein and its application. The amino acid sequence of the heavy chain variable region of the monoclonal antibody to NS2 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 to the non-structural protein NS2 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 NS2; indirect immunofluorescence test shows that it has good reactivity with RSV.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a NS2 protein monoclonal antibody and its application. Background Art

[0002] Human respiratory syncytial virus (RSV) is the most important cause of acute lower respiratory tract diseases in infants around the world. It is reported that approximately 34 million to 64 million children under the age of 5 suffer from bronchiolitis and pneumonia due to RSV infection every year. Infants under 6 months of age have immature immune systems and are more susceptible to infection and even death.

[0003] RSV is an enveloped virus with a non-segmented single-stranded negative-sense RNA genome. It belongs to the genus Orthopneumovirus and its target cells are ciliated bronchial epithelial cells in the human airway. RSV has a diameter of about 150nm and a genome of 15.2kb. It encodes a total of 11 proteins, including 8 structural proteins, 2 nonstructural proteins (NS), and M2-2 whose function has not yet been determined. The 8 structural proteins are nucleocapsid protein, phosphoprotein, matrix protein, small hydrophobic protein, adhesion protein, fusion protein, polymerase, and M2-1.

[0004] Compared with other species in the same family, NS2 is a non-structural protein specific to RSV, with a gene length of 375 bp, encoding 124 amino acid residues. NS2 plays a key role in viral immune evasion, mainly by inhibiting the type I interferon signaling pathway to inhibit the host innate immune response and promote RSV infection. In addition, NS2 inhibits CD8 + T cell response. Other studies have shown that NS1 and NS2 may form a large proteasome-like complex called NSD. In A549 cells, the NSD complex can selectively degrade specific innate immune signaling proteins, such as RIG-I. This complex is metastable, and the stability of NSD can be enhanced through the mitochondrial pathway, while NS2 is mainly localized to mitochondria and can recruit NS1 to mitochondria. In short, a mitochondria-associated NSD complex is formed in RSV-infected cells, thereby selectively degrading innate immune proteins.

[0005] In addition, autophagy in dendritic cells may play a role in RSV infection, and NS2 promotes viral infection through interaction with Beclin-1. NS2 may also promote epithelial cell shedding of RSV-infected ciliated cells in the airway epithelium and enhance the spread of infection by interacting with the cytoskeleton, which may lead to distal airway obstruction, but the exact mechanism by which NS2 protein mediates this process remains to be determined. In summary, NS2 is an antagonist of host interferon and apoptotic responses, and plays a key role in viral replication and host antiviral immunity.

[0006] Since there is currently no commercial anti-RSV NS2 monoclonal antibody (mAb), in-depth research on the function and regulatory mechanism of NS2 protein is limited. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a monoclonal antibody for NS2 protein and its application. The present invention successfully prepares a monoclonal antibody for NS2 protein, and uses the monoclonal antibody for indirect immunofluorescence assay (IFA) and Western blotting detection to analyze the expression and distribution of RSV NS2 in overexpressing cells and virus-infected cells, providing a favorable tool for further studying the interaction mechanism between non-structural protein NS2 and host proteins during RSV infection.

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

[0009] The present invention provides a NS2 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the NS2 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 NS2 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 NS2 protein monoclonal antibody is as shown in SEQ ID No.4.

[0012] Preferably, the CDR sequences of the heavy chain variable regions of the NS2 protein monoclonal antibody are shown in SEQ ID Nos. 5 to 7.

[0013] Preferably, the CDR sequence of the light chain variable region of the NS2 protein monoclonal antibody comprises 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 GTN;

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

[0017] Preferably, the NS2 protein monoclonal antibody is obtained by immunizing animals with RSV NS2 protein.

[0018] Preferably, the amino acid sequence of the RSV NS2 protein is as shown in SEQ ID No.10.

[0019] The present invention also provides the use of the NS2 protein monoclonal antibody described in the above technical solution in the preparation of a drug that specifically binds to RSV NS2 protein.

[0020] The present invention also provides a drug for treating respiratory syncytial virus, comprising the NS2 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 nonstructural protein NS2 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 NS2;

[0025] Indirect immunofluorescence test showed that it had good reactivity with RSV. 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 NS2 protein by SDS-PAGE; M: protein molecular weight standard; 1: precipitate of induced protein after ultrasound; 2: supernatant of induced protein after ultrasound; 3-5: washing solution; 6: eluent; 7: NS2 protein with tag removed;

[0028] Figure 2 Detection of NS2 antibody purification by SDS-PAGE; M: protein molecular weight standard; 1: purified NS2 antibody;

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

[0030] Figure 4 Identification of eukaryotic expressed protein NS2 by Western blotting; 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 The expression and subcellular localization of NS2 protein in transfected cells;

[0032] Figure 6 The expression and subcellular localization of RSV NS2 protein in virus-infected cells. DETAILED DESCRIPTION

[0033] The present invention provides a NS2 protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the NS2 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.

[0034] SEQ ID No.1:

[0035] EVQLQESGPELVRPGTSVNISCKASGSIFTKYWLGWVKQRPGHGLEWIGDIYPGGGYTKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDYAVYFCAREEDFGQGTLVTVSA.

[0036] In the present invention, the nucleotide sequence of the heavy chain variable region of the NS2 protein monoclonal antibody is shown in SEQ ID No.3.

[0037] SEQ ID No.3:

[0038] GAGGTGCAGCTGCAGGAGTCTGGACCTGAGCTGGTAAGGCCTGGGACTTCAGTGAATATATCCTGCAAGGCTTCTGGCTCCATCTTCACTAAATACTGGCTAGGTTGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTACCCTGGAGGTGGTT ATACTAAATACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGTAGCCTGACATCTGAGGACTATGCTGTCTATTTCTGTGCAAGAGAGGAGGATTTTGGCCAAGGGACTCTGGTCACTGTCTCTGCAG.

[0039] SEQ ID No.2:

[0040] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHF.

[0041] In the present invention, the amino acid sequence of the light chain variable region of the NS2 protein monoclonal antibody is shown in SEQ ID No.4.

[0042] SEQ ID No.4:

[0043] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTA ATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTTC.

[0044] In the present invention, the CDR sequences of the heavy chain variable region of the NS2 protein monoclonal antibody are shown in SEQ ID Nos. 5-7.

[0045] SEQ ID No.5:

[0046] GSIFTKYW;

[0047] SEQ ID No.6:

[0048] IYPGGGYT;

[0049] SEQ ID No.7:

[0050] AREED.

[0051] In the present invention, the CDR sequence of the light chain variable region of the NS2 protein monoclonal antibody includes a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence;

[0052] The light chain CDR1 sequence is shown in SEQ ID No. 8, TGAVTTSNY;

[0053] The light chain CDR2 sequence is GTN;

[0054] The light chain CDR3 sequence is shown in SEQ ID No. 9, ALWYSNHF.

[0055] In the present invention, the NS2 protein monoclonal antibody is obtained by immunizing an animal with RSV NS2 protein. The present invention does not specifically limit the method for obtaining the NS2 protein monoclonal antibody by immunizing an animal with RSV NS2 protein, and those skilled in the art can use conventional methods.

[0056] In the present invention, the amino acid sequence of the RSV NS2 protein is shown in SEQ ID No. 10, which is as follows:

[0057] SEQ ID No.10:

[0058] MDTTHNDNTPQRLMITDMRPLSLETIITSLTRDIITHKFIYLINHECIVRKLDERQATFTFLVNYEMKLLHKVGSTKYKKYTEYNTKYGTFPMPIFINHDGFLECIGIKPTKHTPIIYKYDLNP.

[0059] The present invention also provides the use of the NS2 protein monoclonal antibody described in the above technical solution in the preparation of a drug that specifically binds to RSV NS2 protein.

[0060] The present invention also provides a drug for treating respiratory syncytial virus, comprising the NS2 monoclonal antibody described in the above technical solution. In the present invention, the drug preferably also comprises a pharmaceutically acceptable carrier.

[0061] 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.

[0062] The genes, strains, plasmids, viruses and experimental animals used in the following examples are as follows: the sequence of the RSVNS2 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-NS2, pCAGGS-flag-NS2, 293T cells, A549 cells and RSV A2 strains were preserved by 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.

[0063] The main reagents used in the following examples are: restriction endonucleases BamH I and Xho I were purchased from TaKaRa; HA-tagged mouse monoclonal antibody, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, Freund's incomplete adjuvant, Freund's complete adjuvant, fetal bovine serum, Flagbeads and Flagpeptide 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); ECL color development kit was purchased from Merck.

[0064] Example 1

[0065] Construction, expression and purification of RSV NS2 protein

[0066] 1. Construction and identification of NS2 gene prokaryotic expression plasmid

[0067] With reference to the RSV gene published by NCBI (mutant human respiratory syncytial virus strain long, accession number: KF713490.1), the codons of the NS2 full gene were optimized, and restriction sites BamH I and Xho I were introduced at both ends of the expression sequence. The gene sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and constructed into the prokaryotic expression vector pET-32a. The constructed recombinant plasmid was named pET-32a-NS2, and the plasmid was transformed into E. coli DH5α competent cells. After the plasmid was extracted, it was double-digested with BamH I and Xho I for identification, and sequenced and verified by Beijing Qingke Biotechnology Co., Ltd. (Xi'an).

[0068] 2. Prokaryotic expression of NS2 protein

[0069] The recombinant plasmid pET-32a-NS2 was transformed into E. coli BL21 (DE3) competent cells, and the positive monoclonal colonies were picked and inoculated into 5 mL of LB liquid culture medium containing ampicillin (100 μg / mL), and cultured overnight at 37°C and 220 rpm. The glycerol bacteria were preserved, and the bacterial liquid was inoculated into 100 mL of LB liquid culture medium containing ampicillin 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 8000 rpm 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 7.0), and ultrasonically disrupted. The supernatant and precipitate were collected separately, and the precipitate was resuspended and dissolved with 8 M urea. After the sample was prepared, the expression of NS2 protein was observed by SDS-PAGE electrophoresis.

[0070] 3. Purification of NS2 Protein

[0071] The stored glycerol bacteria were induced to express in large quantities according to the above steps, the cells were collected by centrifugation, and the cell supernatant was collected by centrifugation after ultrasonic disruption. The nickel column was connected to the AKTApure 150 purifier, and the collected supernatant was filtered through a 0.45 μm filter membrane and purified by affinity chromatography technology. The impurities were washed and eluted with a linear gradient imidazole buffer in turn, and the washing liquid and the elution liquid were collected respectively. The purified fusion protein was digested with thrombin at room temperature for 3 hours, and further purified by affinity chromatography technology to remove the thioredoxin tag, and the protein purification was detected by SDS-PAGE.

[0072] According to the affinity chromatography purification method, the RSV NS2 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 , 124 amino acid residues, a protein size of 14.7 kDa, an isoelectric point of 8.74, and its amino acid sequence is shown in SEQ ID No. 10, as follows:

[0073] MDTTHNDNTPQRLMITDMRPLSLETIITSLTRDIITHKFIYLINHECIVRKLDERQATFTFLVNYEMKLLHKVGSTKYKKYTEYNTKYGTFPMPIFINHDGFLECIGIKPTKHTPIIYKYDLNP.

[0074] Example 2

[0075] Preparation of monoclonal antibodies

[0076] 1. Immunization of mice with purified NS1 protein

[0077] To prepare monoclonal antibodies against RSV NS1 protein, the purified NS1 protein was emulsified with an adjuvant at a ratio of 1:1 to prepare a vaccine, and six female BALB / c mice aged 6-8 weeks were immunized. The first immunization was emulsified with Freund's complete adjuvant, and the second to fourth immunizations were emulsified with Freund's incomplete adjuvant. Immunizations were performed on days 0, 14, 28, and 36, respectively, with an amount of 50 μg / mouse protein each time. 14 days after the fourth immunization, the mice were tail-cut and blood was collected, and the serum was separated and the antibody titer was determined.

[0078] 2. Evaluation of mouse serum titer by indirect ELISA

[0079] RSV NS1 protein was used as the coating antigen, the antigen was diluted with coating buffer, added to the ELISA microplate, 100 ng / well, 100 μL / well, and left to stand overnight at 4°C. The ELISA reaction plate coated with the antigen was removed, the liquid was discarded, the plate was washed 5 times with 1×PBST, and patted dry. 2% bovine serum albumin (BSA) was added, 200 μL / well, blocked at 37°C for 2h, washed 5 times with 1×PBST and patted dry. SP2 / 0 prepared mouse ascites was used as a negative control, mouse serum (starting from 1:1000, 2-fold gradient dilution) was used as the primary antibody, 2-fold gradient dilution was performed with 1×PBST (starting from 1:1000), and added to the ELISA reaction plate, 100 μL / well. Incubate at 37°C for 1h, discard the liquid, wash the plate 5 times with 1×PBST and pat dry. Dilute HRP-labeled goat anti-mouse IgG with 1×PBST (1:10000) and add to each well, 100 μL / well, incubate at 37°C for 1 hour, discard the solution, wash the plate 5 times and pat dry. Add TMB substrate colorimetric solution, 50 μL / well, incubate at 37°C in the dark for 15 minutes. Add stop solution, 50 μL / well, and measure the OD450 value on the microplate reader within 5 minutes. 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.

[0080] 3. Screening of hybridoma cells

[0081] 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 the HRP-labeled goat anti-mouse IgG antibody as the secondary antibody to screen the positive hybridoma cell lines that reacted with RSVNS1. The positive hybridoma cell lines were cloned and purified three times and then subcultured and expanded. The cells were collected and 2×10 6 Cells / branch were stored in liquid nitrogen.

[0082] 4. Preparation and purification of monoclonal antibodies

[0083] One week before the injection of monoclonal cell lines, sterilized 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 12000rpm for 10 minutes, and impurities such as lipids and tissue fragments were removed by filtration to obtain clear ascites. The antibodies were purified by affinity chromatography. The purification method is as follows:

[0084] (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

[0085] (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.

[0086] (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.

[0087] (4) Washing: After loading, wash the column with 5-10 times the column volume of equilibration buffer and collect the effluent.

[0088] (5) Elution: Use 5-10 times elution buffer (0.1 M glycine, pH 3.0) to elute and collect the effluent.

[0089] (6) pH adjustment: After elution, the pH of the eluted product was immediately adjusted to neutral with saturated sodium carbonate.

[0090] (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.

[0091] (8) Storage of chromatography columns: After flushing the affinity chromatography column with 5 column volumes of 20% ethanol, store at 4°C.

[0092] (9) Sample concentration: Use a 10 kDa ultrafiltration tube to concentrate the eluted product to about 1-5 mL.

[0093] (10) Dialysis: The concentrated sample was placed in a dialysis bag and dialyzed overnight in 5 L buffer (0.01 M PBS, pH = 7.4). The solution was changed once on the second day.

[0094] (12) Antibody purity was analyzed by SDS-PAGE and protein content was determined.

[0095] The results of SDS-PAGE analysis are shown in Figure 2 The antibody heavy chain and light chain bands were shown at 50kDa and 25kDa, respectively. The content of the purified NS2 monoclonal antibody was 2mg / mL.

[0096] (13) Western blotting results ( Figure 3 ) showed that the screened NS2 monoclonal antibody had good reactivity with the purified NS2 protein.

[0097] 5. Monoclonal Antibody Subtype Identification and Sequencing

[0098] (1) The monoclonal antibody Ig subtype was identified using a mouse monoclonal antibody subtype identification kit (Proteintech), and the NS2 monoclonal antibody subtype IgG1 was measured.

[0099] (2) Recovery of hybridoma cells: Take out a hybridoma cell cryopreservation tube containing a specific monoclonal antibody against the NS2 protein of respiratory syncytial virus 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, transfer the cell fluid into 10 mL of RPMI-1640 culture medium, and 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.

[0100] (3) PCR amplification of the variable region of the monoclonal antibody: The cells were collected, 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 ScriptRTMaxMasterMix. The prepared cDNA was used as a template and its gene fragment was amplified using a high-fidelity polymerase. The system was 2×RapidTaqMasterMix 25 μL, 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, 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.

[0101] (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 and analyzed 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 NS2 protein monoclonal antibody prepared in this example are shown in Table 1.

[0102] Table 1 Antibody variable region sequences

[0103] CDR1 CDR2 CDR3 Heavy chain variable region GSIFTKYW IYPGGGYT AREED Light chain variable region TGAVTTSNY GTN ALWYSNHF

[0104] Among them, the encoding heavy chain amino acid sequence is (SEQ ID No.1):

[0105] EVQLQESGPELVRPGTSVNISCKASGSIFTKYWLGWVKQRPGHGLEWIGDIYPGGGYTKYNEKFKGKATLTADTSSSTAYMQLSSLTSEDYAVYFCAREEDFGQGTLVTVSA.

[0106] The gene sequence encoding the heavy chain variable region is (SEQ ID No.3):

[0107] GAGGTGCAGCTGCAGGAGTCTGGACCTGAGCTGGTAAGGCCTGGGACTTCAGTGAATATATCCTGCAAGGCTTCTGGCTCCATCTTCACTAAATACTGGCTAGGTTGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTACCCTGGAGGTGGTT ATACTAAATACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACACATCCTCCAGCACTGCCTACATGCAGCTCAGTAGCCTGACATCTGAGGACTATGCTGTCTATTTCTGTGCAAGAGAGGAGGATTTTGGCCAAGGGACTCTGGTCACTGTCTCTGCAG.

[0108] The amino acid sequence encoding the light chain variable region is (SEQ ID No.2):

[0109] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHF.

[0110] The gene sequence encoding the light chain variable region is (SEQ ID No.4):

[0111] CAGGCTGTTGTGACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTA ATAGGTGGTACCAACAACCGAGCTCCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTTC.

[0112] Example 3

[0113] Monoclonal antibody specifically reacts with nonstructural protein NS2

[0114] Western blotting to detect the expression of NS2 protein in overexpressing cells

[0115] The eukaryotic expression plasmid pCAGGS-HA-NS2 and the empty plasmid pCAGGS-HA were transfected into 293T cells, respectively. The cell samples were collected and processed, and their reactivity with NS2 monoclonal antibody was detected by Western blotting. The specific experimental process is as follows:

[0116] (1) The eukaryotic expression plasmid pCAGGS-HA-NS2 and the empty plasmid pCAGGS-HA were transfected into 293T cells, respectively, and the 293T cells were collected after culturing at 37°C for 24 h.

[0117] (2) Sample preparation: Lyse cells with 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.

[0118] (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 of sample 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 bromophenol blue reaches the bottom of the separation gel, then terminate the electrophoresis.

[0119] (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 the PVDF membrane to the same size as the gel and soak it in 100% methanol for 1-2 minutes. 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, put them in the wet transfer tank, add enough transfer buffer, and transfer the membrane for 2 hours at a constant voltage of 100V.

[0120] (5) Blocking: After transfer, remove the PVDF membrane and block it with 5% skimmed milk powder at room temperature for 1-2 h.

[0121] (6) Incubation with primary antibody: Add 5 mL of anti-NS2 mouse monoclonal antibody diluted in 5% skim milk powder (1:1000) and incubate at 4°C overnight.

[0122] (7) Incubation with secondary antibody: Wash with 1× PBST five times, shake for 5 min each time at room temperature, add 5 mL of horseradish peroxidase-labeled goat anti-mouse IgG antibody diluted with 5% skim milk powder (1:5000), and incubate at room temperature for 1 h.

[0123] (8) Development: Wash with 1× PBST five times, shaking for 5 min each time under room temperature conditions, mix ECL substrate color reagent A and reagent B in a 1:1 ratio, add an appropriate amount of color developing solution to the PVDF membrane, scan and expose, and save the image.

[0124] Test results are shown in Figure 4 The NS2 monoclonal antibody of the present invention specifically reacts with the RSV non-structural protein NS2, but not with the RSV non-structural protein NS1 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 NS2 protein. This indicates that the monoclonal antibody of the present invention can specifically react with the RSV non-structural protein NS2 protein.

[0125] Example 4

[0126] Determination of the Reactivity of Monoclonal Antibodies to RSV Nonstructural Protein NS2

[0127] 1 Immunofluorescence detection of NS2 protein expression and subcellular localization analysis in overexpressed cells

[0128] A549 cells were cultured at 1×10 5 / well were inoculated in a confocal dish, and the cells were transfected with eukaryotic expression plasmid pCAGGS-HA-NS2 and empty plasmid pCAGGS-HA for 24 hours. The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS for 4 times, and permeabilized with 0.2% TritonX-100 in PBS solution at room temperature for 10 minutes; the solution was discarded, and the cells were blocked with Blocking buffer solution containing 2% BSA at room temperature for 1 hour; RSVNS2 monoclonal antibody was diluted with the above Blocking buffer solution, and the primary antibody was incubated at 4°C 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°C in the dark for 1 hour; PBS was washed 4 times, DAPI was diluted with PBS, and the nucleus was stained for 5 minutes at room temperature in the dark, and PBS was washed 4 times, and the images were observed under a confocal microscope. Confocal results ( Figure 5 ) showed that the monoclonal antibody bound well to the transient expression product of RSV NS2 in A549 cells with strong specificity. No fluorescence was presented in the empty vector transfection control group. In cells transfected with pCAGGS-HA-NS2, the NS2 protein was mainly distributed in the cytoplasm, with a small amount localized in the nucleus, and formed dot-like aggregates around the nucleus, indicating that the monoclonal antibody described in the present invention has good reactivity with the RSV non-structural protein NS2 protein.

[0129] 2 Immunofluorescence detection of NS2 protein expression and subcellular localization analysis in RSV-infected cells

[0130] A549 cells were adsorbed with RSV MOI=1 virus for 2 hours, and uninfected cells were used as negative controls. The confocal dish was gently shaken every 30 minutes. After 2 hours of adsorption, the cells were cultured with complete medium containing 10% fetal bovine serum for 48 hours. The cells were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS for 4 times, and permeabilized with 0.2% TritonX-100 in PBS solution at room temperature for 10 minutes; the solution was discarded, and the cells were blocked with Blocking buffer solution containing 2% BSA at room temperature for 1 hour; RSV NS2 monoclonal antibody was diluted with the above Blocking buffer solution, and the primary antibody was incubated at 4°C overnight; PBS was washed 4 times, and the goat anti-mouse 488 green fluorescent secondary antibody was diluted with PBS, and incubated in a constant temperature incubator at 37°C in the dark for 1 hour; PBS was washed 4 times, and the nuclei were stained with DAPI at room temperature in the dark for 5 minutes, and PBS was washed 4 times, and the cells were observed and photographed under a confocal microscope. Results ( Figure 6) showed that the NS2 monoclonal antibody had a good specific reaction with RSV-infected A2549 cells and had no non-specific binding in uninfected A549 cells. In addition, the NS2 protein was mainly distributed in the cytoplasm in RSV-infected cells, with a small amount localized in the nucleus and forming dot-like aggregates around the nucleus, indicating that the monoclonal antibody described in the present invention has a good reactivity with the NS2 protein of the RSV A2 strain.

[0131] 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 NS2 protein, characterized in that: The amino acid sequence of the heavy chain variable region of the RSV NS2 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. The RSV NS2 protein monoclonal antibody according to claim 1, characterized in that The nucleotide sequence of the heavy chain variable region of the RSV NS2 protein monoclonal antibody is shown in SEQ ID No.

3.

3. The RSV NS2 protein monoclonal antibody according to claim 1, characterized in that The nucleotide sequence of the light chain variable region of the RSV NS2 protein monoclonal antibody is shown in SEQ ID No.

4.

4. The RSV NS2 protein monoclonal antibody according to claim 1, characterized in that The CDR sequences of the heavy chain variable regions of the RSV NS2 protein monoclonal antibodies are shown in SEQ ID Nos. 5-7.

5. The RSV NS2 protein monoclonal antibody according to claim 1, characterized in that The CDR sequence of the light chain variable region of the RSV NS2 protein monoclonal antibody consists of a light chain CDR1 sequence, a light chain CDR2 sequence and a light chain CDR3 sequence; The light chain CDR1 sequence is shown in SEQ ID No.8; The light chain CDR2 sequence is GTN; The light chain CDR3 sequence is shown as SEQ ID No.

9.

6. The RSV NS2 protein monoclonal antibody according to claim 1, characterized in that The RSV NS2 protein monoclonal antibody is obtained by immunizing animals with RSV NS2 protein.

7. The RSV NS2 protein monoclonal antibody according to claim 6, characterized in that The amino acid sequence of the RSV NS2 protein is shown in SEQ ID No.

10.

8. Use of the RSV NS2 protein monoclonal antibody according to any one of claims 1 to 7 in the preparation of a medicament for treating respiratory syncytial virus infection.

9. A drug for treating respiratory syncytial virus infection, characterized in that: The invention comprises the RSV NS2 protein monoclonal antibody according to any one of claims 1 to 7.

10. The drug according to claim 9, characterized in that Also included are pharmaceutically acceptable carriers.

Images