Anti-varicella-zoster virus monoclonal antibody and its application

Abstract

The present invention provides an anti-varicella-zoster virus monoclonal antibody and its application, and relates to the technical field of monoclonal antibody screening and preparation, and more specifically, a method of immunizing mice with a recombinant varicella-zoster virus gE trimer protein vaccine, and screening and preparing anti-varicella-zoster virus monoclonal antibodies by hybridoma method, and the specific application of the antibody. The recombinant VZV-gE trimer protein vaccine of the present invention has good immunogenicity and can induce higher immune serum titers. The human-mouse chimeric monoclonal antibody of the present invention has good binding activity and affinity to VZV-gE protein, and the indirect ELISA method for detecting gE protein has good linear relationship and detection limit, and is expected to be used for VZV vaccine evaluation and VZV infection diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, and in particular to an anti-varicella-zoster virus monoclonal antibody and an application thereof. Background Art

[0002] Varicella-zostervirus (VZV) is a highly contagious alpha-herpes virus, an enveloped linear double-stranded DNA virus with only one serotype. The VZV genome has 71 genes, encoding 67 different proteins, including 6 glycoproteins (gE, gB, gH, gI, gC and gL). Among them, VZV-gE protein is the most highly expressed glycoprotein of VZV and has multiple functions. First, it is the attachment protein of the virus, which can bind to the receptors of the host cell, thereby mediating the entry of the virus into the host cell. Secondly, the gE protein is also involved in the assembly and release of the virus. In addition, it can also regulate the latent process of the virus in the ganglion.

[0003] VZV is mainly transmitted through air droplets, direct contact, and vertical transmission from mother to child. After the virus enters the respiratory tract, it replicates and proliferates in the lymph nodes near the upper respiratory tract. During the initial viremia, the virus is released from the lymph nodes and enters the blood, causing the initial viremia. At this time, the virus will spread to multiple organs throughout the body, including the liver and spleen. During the secondary viremia, the virus spreads to the skin and mucous membranes with the blood. At this time, the characteristic rash begins to appear, initially erythema, and quickly develops into fluid-filled blisters. After infection, the virus lurks in the cranial nerves and dorsal root ganglia of the spinal cord for a long time. When immunity decreases, the virus can be reactivated, causing localized herpes zoster (along the distribution area of ​​sensory nerves) or causing systemic dissemination in immunocompromised individuals. In recent years, the incidence of varicella and herpes zoster in my country has shown an upward trend. According to statistics, varicella and herpes zoster have caused a considerable disease burden worldwide, with at least 140 million new cases and 4,200 deaths each year.

[0004] Recombinant protein vaccines have certain advantages in safety because they do not contain live viruses or complete components of the virus. At the same time, recombinant trimer antigens refer to the trimer structure of virus or pathogen surface proteins expressed in vitro through recombinant DNA technology, which has the characteristics of good stability, high immunogenicity and strong safety. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-varicella-zoster virus monoclonal antibody and its application. The recombinant varicella-zoster virus glycoprotein E trimer protein was used to immunize mice, and the antibody titer of mouse serum was increased compared with the monomeric recombinant varicella-zoster virus glycoprotein E. Furthermore, a highly active anti-VZV-gE protein monoclonal antibody was sorted by hybridoma technology, which can effectively bind to the VZV-gE protein (named VD460-1).

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant VZV-gE trimer protein, the amino acid sequence of the recombinant VZV-gE trimer protein is shown in SEQ ID NO.5.

[0008] The present invention also provides the use of the recombinant VZV-gE trimer protein in preparing a monoclonal antibody against the VZV-gE protein.

[0009] The present invention also provides an antibody prepared using the above recombinant VZV-gE trimer protein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are respectively as shown in the amino acid sequences at positions 26-33, 51-58 and 97-107 of SEQ ID NO.1;

[0010] The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are shown in the amino acid sequences at positions 27-36, 54-56 and 93-100 of SEQ ID NO.2, respectively.

[0011] The present invention also provides a gene encoding the above antibody, comprising a VD460-1H nucleotide sequence as shown in SEQ ID NO.3 and a VD460-1L nucleotide sequence as shown in SEQ ID NO.4.

[0012] The present invention also provides an antigen binding fragment that binds to VZV-gE antigen protein, comprising VH and VL;

[0013] The VH comprises amino acids 1 to 118 of the amino acid sequence shown in SEQ ID NO.1;

[0014] The VL comprises amino acids 1 to 110 having an amino acid sequence as shown in SEQ ID NO.2.

[0015] The present invention also provides the use of the above antibody, the above encoding gene or the above antigen binding fragment in preparing a reagent or a kit for detecting VZV-gE protein.

[0016] The present invention also provides the use of the above antibody, the above encoding gene or the above antigen binding fragment in preparing a reagent or a kit for detecting herpes zoster virus.

[0017] The present invention also provides the use of the above antibody, the above encoding gene or the above antigen binding fragment in the preparation of herpes zoster virus vaccine.

[0018] The present invention also provides the use of the above-mentioned antibody, the above-mentioned encoding gene or the above-mentioned antigen-binding fragment in the preparation of VZV vaccine evaluation products.

[0019] The present invention provides a reagent or a kit for detecting herpes zoster virus, comprising the above antibody or the above antigen binding fragment.

[0020] Beneficial effects of the present invention:

[0021] 1. The monoclonal antibody of the present invention has good binding activity to VZV-gE protein and is expected to be used for VZV vaccine evaluation and VZV infection diagnosis. The antibody has high affinity and is located on the VZV-gE protein. Therefore, these antibodies can be used as competitive antibodies, indirect ELISA methods and double-antibody sandwich ELISA detection antibodies, and are used to measure neutralizing antibody titers after VZV vaccination or evaluate vaccine immunogenicity. VD460-1 can effectively bind to VZV-gE protein, and the EC50 is 147ng / mL. VD460-1 has a high affinity for VZV-gE protein, and the Kd value is 13.16nM. The minimum detection concentration of the indirect ELISA method for monomeric VZV-gE protein is 15.63ng / mL; the minimum detection concentration of the indirect ELISA method for recombinant VZV-gE trimer protein is 1.95ng / mL.

[0022] 2. The recombinant VZV-gE trimeric protein vaccine of the present invention is a trimer, and each product molecule contains 3 VZV-gE proteins, has good immunogenicity, and can overcome the problems of low efficiency of original VZV-gE protein expression and purification and low titer of immune serum.

[0023] 3. In order to make the expressed trimeric protein more stable, the present invention optimizes and displays the optimal GS-Linker length and sequence before and after the trimerization domain of the recombinant VZV-gE trimeric protein T4 bacteriophage fiber protein, and the purity of the obtained product is above 99%.

[0024] 4. In order to increase the yield of the trimer protein, the present invention replaces the recombinant VZV-gE trimer protein signal peptide with a tissue-type plasminogen activator signal peptide, and the expression and purification yield can reach 100 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1This is a schematic diagram of the sequence structure of the recombinant VZV-gE trimer protein of the present invention;

[0026] Figure 2 is the SDS-PAGE pattern of the recombinant VZV-gE trimer protein of the present invention;

[0027] Figure 3 Comparison of the titer of the first immune serum of mice injected with the recombinant VZV-gE trimer protein of the present invention and the VZV-gE monomer protein;

[0028] Figure 4 Comparison of the titer of the second shot of immune serum of mice with the recombinant VZV-gE trimer protein of the present invention and the VZV-gE monomer protein;

[0029] Figure 5 The binding activity of the five candidate antibodies provided in the embodiments of the present invention against the VZV-gE protein;

[0030] Figure 6 The SDS-PAGE pattern of the purified monoclonal antibody of the present invention;

[0031] Figure 7 The verification provided in the embodiments of the present invention is for the binding activity of VD460-1 against VZV-gE protein;

[0032] Figure 8 The affinity of VD460-1 for VZV-gE protein provided in the embodiments of the present invention is verified;

[0033] Fig. 9 The linear range of VD460-1 provided in the embodiment of the present invention for VZV-gE protein detection;

[0034] Fig.10 This is the linear range of VD460-1 provided in the embodiment of the present invention for detecting recombinant VZV-gE trimer protein. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] In the embodiments of the present invention, the sequences of SEQ ID NO.1 to SEQ ID NO.5 are respectively as follows:

[0037] SEQ ID NO.1

[0038] > VD460-1H amino acid sequence

[0039] EVQLQQSGAELVKPGASVKLSCTASVFNIKDTYMYWVNQRPEQGLEWIGRIDPVNGNTKYDPKFQDKATITSDTSSNTAYLQLSSLTSEDTAVYYCAKEDWDGGLLAGARGLWSLSLQASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0040] SEQ ID NO.2

[0041] > Amino acid sequence of VD460-1L

[0042] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0043] SEQ ID NO.3

[0044] > Nucleotide sequence of VD460-1H

[0045]

[0046] SEQ ID NO.4

[0047] > VD460-1L nucleotide sequence

[0048] GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTtACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGAGAATGC

[0049] SEQ ID NO.5

[0050] > Amino acid sequence of recombinant VZV-gE trimer protein

[0051] MDAMKRGLCCVLLLCGAVFVSPRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQ RQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWN MRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFG LILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRGSGGGGSGGGGSGGGGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGSGSGSGSGGIEGRHHHHHH Example 1

[0052] Preparation of recombinant VZV-gE trimer protein:

[0053] 1. Recombinant protein codon optimization: Optimize the nucleotide sequence of the recombinant protein codon, including: 1. Avoid commonly used restriction sites; 2. Optimize according to the codon preference of 293F cell transfection expression; 3. To improve transcription efficiency, avoid too high or too low GC content and keep it at 40%-60%. The schematic diagram of the recombinant VZV-gE protein sequence structure is shown in Figure 1 After optimization, plasmid synthesis was performed on the pcDNA3.1 vector to obtain the D3VD460 expression vector, the specific sequence of which is shown in SEQ ID NO.5.

[0054] 2. Plasmid transfection: After obtaining the D3VD460 expression vector, D3VD460 was mixed and co-transfected into 100 mL of 293F cells to express the recombinant VZV-gE protein. The cell culture fluid was collected after 6 days.

[0055] 3. Culture medium pretreatment: After the cell culture medium was centrifuged at 4800 rpm for 10 min in a horizontal centrifuge, the supernatant was filtered using a 0.45 μm filter to obtain the filtered culture medium.

[0056] 4. Purification: Use 20mM phosphate and 0.5M NaCl to make a balance solution with pH=7.4; use 20mM phosphate, 0.5M NaCl and 5mM imidazole to make a wash solution with pH=7.4; use 20mM phosphate, 0.5M NaCl and 250mM imidazole to make an eluent with pH=7.4. Use AKTA purification chromatography system for purification. First, use the balance solution to level the His affinity chromatography column, then pass the sample through the column, wash with the wash solution until the UV peak is level, then elute, and collect the eluent.

[0057] 5. Concentration: After purification, ultrafiltration was performed using a 10k ultrafiltration column, and ultrafiltration was performed using PBS for 2 times, and the ultrafiltrate was collected.

[0058] 6. Enzyme digestion: After ultrafiltration, mix the mixture in a ratio of Xa protease: fusion protein = 1:100 and incubate at 4°C for 24 hours.

[0059] 7. Tag removal: Re-purify according to the above purification steps and collect the puncture fluid.

[0060] 8. Ultrafiltration: After purification, use a 10k ultrafiltration column to ultrafilter the puncture liquid, and use PBS ultrafiltration replacement twice to collect the ultrafiltrate.

[0061] Results: SDS-PAGE was used to detect the recombinant VZV-gE trimer protein and VZV-gE protein monomer after affinity chromatography purification. For details, see Figure 2 A, lane M is a molecular weight marker; lane Ctr is a negative control; lane gE-Tri is a recombinant VZV-gE trimer protein. Figure 2 B, lane M is a molecular weight marker; lane gE-Mo is a recombinant VZV-gE monomer protein; lane gE-Tri is a recombinant VZV-gE trimer protein. Example 2

[0062] Mouse immunization and serum titer determination

[0063] 1. Mouse immunization: 6-week-old female Kunming mice were immunized with recombinant VZV-gE trimer protein. The mice were randomly divided into 2 groups, 6 in each group. Group 1 used VZV-gE monomer protein, and Group 2 used recombinant varicella-zoster virus glycoprotein E trimer protein. All groups were mixed with aluminum adjuvant in a 1:1 ratio to a final volume of 100 μL. Subcutaneous immunization was performed at week 0 and week 3, with a dose of 5 μg per mouse. Blood was collected by cutting the tail on the 14th day after each immunization. Intraperitoneal booster immunization was performed on the 4th day of the 6th week, with 5 μg per mouse. The spleen was taken for hybridoma fusion at week 7.

[0064] 2. Serum titer determination

[0065] 2.1 Coating antigen: Dilute VZV-gE protein to 1 μg / mL, spread 100 μL / well on a 96-well ELISA plate, and incubate at 4°C overnight.

[0066] 2.2 Blocking: Wash the plate three times with PBST solution, add 100 μL of 5% skimmed milk powder to each well and block at 37°C for 1.5 h.

[0067] 2.3 Sample incubation: Wash the plate 5 times with PBST solution, dilute the serum sample to 1:100 first, then dilute it 3-fold in 8 wells, add 100 μL of the diluted sample to each well, and block at 37°C for 1 h.

[0068] 2.4 Secondary antibody incubation: Wash the plate 5 times with PBST buffer, dilute the secondary antibody to 2.5 μg / mL, add 100 μL of the diluted secondary antibody to each well, and block at 37°C for 1 hour.

[0069] 2.5 Color development: Wash the plate 5 times with PBST solution, add 100 μL TMB color development solution to each well, develop color for 15 minutes at room temperature away from light, then add 50 μL ELISA stop solution to each well to stop color development.

[0070] 2.6 Reading: Detect the OD value of each well at 450-630 nm on a microplate reader, draw the curve using the Logistic four-parameter fitting method, and calculate the EC50 value of the antibody.

[0071] Results: The serum titer of mice in the recombinant VZV-gE trimer vaccine group was higher than that in the VZV-gE monomer protein vaccine group 14 days after the first and second injections (P<0.05). Figure 3 , 4 . Example 3

[0072] Gene extraction from dominant cell lines

[0073] 1. Cell fusion: Blood was collected from the eyeballs of mice 3 days after booster immunization, and then the mice were killed by dislocating the neck. After disinfection, the spleen was taken out in the clean bench, and the spleen was ground in a 200-mesh sieve. Medium B was added to 5 mL, and the final volume of all spleens was 30 mL. After centrifugation at 400g for 5 minutes, the supernatant was discarded, 30 mL of red blood cell lysis buffer was added, and lysis was performed on ice for 5 minutes. After shaking, 20 mL of PBS was added to stop, and centrifugation at 400g for 5 minutes. After the SP2 / 0 cells were blown away, the SP2 / 0 cells and spleen cells were counted separately. SP2 / 0 cells and spleen cells were mixed in a ratio of 1:2. After full fusion, the two were fused using the Demo18 ModelEX electric fusion instrument. After fusion, the hybridoma cells were left to stand for 5 minutes to obtain hybridoma cells, which were placed in a large dish containing 15 mL of MediumC, mixed, and cultured in a 37°C 5% CO2 incubator for 24 hours.

[0074] 2. Cell screening and culture: Blow off the cells in the large dish and centrifuge at 400g for 5 minutes. Use HAT medium to disperse the cells and evenly distribute them to 96-well plates, 100μL per well. After the cells have grown all over the bottom of the plate, aspirate the supernatant, use indirect ELISA to detect the binding activity of the supernatant with VZV-gE, select the wells with high binding activity, discard the supernatant, and transfer to a 24-well plate for culture with Medium E. After the cells have grown all over the bottom of the plate, aspirate the supernatant, use indirect ELISA to detect the binding activity of the supernatant with VZV-gE, select the wells with high binding activity and evenly distribute them to 96-well plates for subcloning, and repeat the above steps 3 times.

[0075] 3. Indirect ELISA method to detect supernatant:

[0076] 3.1 Coating antigen: Dilute VZV-gE protein to 1 μg / mL, spread 100 μL / well on a 96-well ELISA plate, and incubate at 4°C overnight.

[0077] 3.2 Blocking: Wash the plate three times with PBST solution, add 100 μL of 5% BSA solution to each well, and block at 37°C for 1.5 h.

[0078] 3.3 Sample incubation: Wash the plate 5 times with PBST solution, aspirate 11 μL of culture supernatant from each well, mix with 99 μL PBS, then add 100 μL of diluted sample to each well in turn, and block at 37°C for 1 hour.

[0079] 3.4 Secondary antibody incubation: Wash the plate 5 times with PBST buffer, dilute the secondary antibody to 2.5 μg / mL, add 100 μL of the diluted secondary antibody to each well, and block at 37°C for 1 hour.

[0080] 3.5 Color development: Wash the plate 5 times with PBST solution, add 100 μL TMB color development solution to each well, develop color for 15 minutes at room temperature away from light, then add 50 μL ELISA stop solution to each well to stop color development.

[0081] 3.6 Reading: Detect the OD value of each well at 450-630nm on a microplate reader, draw the curve using the Logistic four-parameter fitting method, and calculate the EC50 value of the antibody.

[0082] Results: Five superior cell lines with high binding activity were screened: VD460-1, G5, B5, D1, and F7. Example 4

[0083] Purification and identification of culture supernatant of dominant cell lines

[0084] 1. Purification of culture supernatant

[0085] 1.1 Culture medium pretreatment: After the cell culture medium was centrifuged at 4800 rpm for 10 min in a horizontal centrifuge, the supernatant was filtered using a 0.45 μm filter to obtain the filtered culture medium.

[0086] 1.2 Purification and incubation: Use 0.15M NaCl and 20mM Na2HPO4 to make 10xBufferA solution, add 10xBuffer to the filtered culture fluid at a ratio of filtered culture fluid: 10xBuffer = 9:1, then add 50μL ProteinA filler to the filtered culture fluid, and incubate at 25℃ for 2h.

[0087] 1.3 Purification: Assemble the gravity column, wet the filter pad and add 200uL ProteinA filler, dilute 10x Buffer to 1x and pass 100μL of the column to balance the filler, add the incubated sample after equilibration and pass the column twice, then elute 100μL with 1x Buffer, use 4mL 0.1M glycine for elution, and add 440μL Tris NaCl after elution.

[0088] 1.4 Ultrafiltration: After purification, use a 10k ultrafiltration column for ultrafiltration, and use PBS for ultrafiltration replacement twice to collect the ultrafiltrate.

[0089] 2. Identification of Binding Ability of Purified Antibodies

[0090] 2.1 Coating antigen: Dilute VZV-gE protein to 2 μg / mL, plate 100 μL / mL well on a 96-well ELISA plate, and incubate at 4°C overnight.

[0091] 2.2 Blocking: Wash the plate three times with PBST solution, add 100 μL of 5% BSA solution to each well, and block at 37°C for 1.5 h.

[0092] 2.3 Sample incubation: Wash the plate 5 times with PBST solution, dilute the 5 candidate antibodies to 10 μg / mL respectively, and then add 100 μL of the diluted sample to each well in turn, and block at 37°C for 1 hour.

[0093] 2.4 Secondary antibody incubation: Wash the plate 5 times with PBST buffer, dilute the secondary antibody to 2.5 μg / mL, add 100 μL of the diluted secondary antibody to each well, and block at 37°C for 1 h.

[0094] 2.5 Color development: Wash the plate 5 times with PBST solution, add 100 μL TMB color development solution to each well, develop color for 15 minutes at room temperature away from light, then add 50 μL ELISA stop solution to each well to stop color development.

[0095] 2.6 Reading: Detect the OD value of each well at 450-630 nm on a microplate reader, draw the curve using the Logistic four-parameter fitting method, and calculate the EC50 value of the antibody.

[0096] Results: The binding activity of the five candidate antibodies was Figure 5 As shown, the EC50 value of monoclonal antibody VD460-1 is the highest, see Figure 5 . Example 5

[0097] Gene extraction of monoclonal antibody VD460-1 and human-mouse chimera transformation

[0098] 1. Gene extraction of dominant cell lines: Blow the dominant cell lines off the cell culture dish, centrifuge the cells at 4800rpm for 2min, discard the supernatant, add 500μL of PBS solution to each tube, blow the cells apart, centrifuge at 4800rpm for 2min, and repeat the wash. Use an RNA extraction kit to extract total cell RNA. Total RNA is reverse transcribed into cDNA, and then the antibody V region sequence in the total cDNA is amplified using mouse antibody light and heavy chain specific primers. After amplification, add A ends to both ends of the V region fragment according to the kit. Then use the kit to connect it to the T vector. Transform into the competent state, add anti-free culture medium after heat machine and culture for 1h. Mix XGal and IPTG at a ratio of 1:2 and apply 90μL on a solid culture medium containing ampicillin, apply 100μL of bacterial solution to the culture medium, and culture for 16h. After cultivation, select the larger and brighter monoclonal colonies with white color, culture them in 800μL ampicillin culture medium for 10h and then sequence them.

[0099] 2. Amplify the constant region genes of light and heavy chains and human-mouse chimeric monoclonal antibodies: After sequencing, synthesize primers at the C-terminus and N-terminus of the light and heavy chain variable regions, and use FLASH PCR amplification enzyme to amplify the antibody VH and VL regions after synthesis.

[0100] 3. Agarose gel electrophoresis: Add 5.5 μL of 5x Loading Burrfer to the PCR reaction product, and perform electrophoresis on 1.2% agarose gel at 150 V for 27 min. After electrophoresis, cut out the target band under a 320 nm light source, and recover VH, VL, and fragments carrying Fc and light chain constant regions using the DNA gel recovery kit.

[0101] 4. Homologous recombination: Calculate the recombination system according to the formula: vector mass (50-100ng) / vector length (bp)*fragment length (bp)*multiple = xng (fragment mass), use SOSO homologous recombination enzyme to recombinant at 50°C for 20min, transform to competent state, add antibiotic-free medium for 1h after heat machine, centrifuge at 400g for 2min, resuspend the bacterial solution in 100μL antibiotic-free medium, apply it to solid medium containing ampicillin, and culture for 16h. After picking bacteria on the second day, culture for 10h and sequence. After sequencing, the correct sequence is compared and extracted from the plasmid to obtain VD460-1H and VD460-1L expression vectors.

[0102] Results: VD460-1H and VD460-1L expression vectors were obtained, the specific amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, and the specific nucleotide sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. Example 6

[0103] Transfection, expression and purification of monoclonal antibody VD460-1

[0104] 1. Plasmid transfection: After obtaining the VD460-1H and VD460-1L expression vectors, VD460-1H and VD460-1L were mixed and co-transfected into 100 mL of 293F cells to express the monoclonal antibody D460-1. The cell culture fluid was collected after 6 days.

[0105] 2. Culture medium pretreatment: After the cell culture medium was centrifuged at 4800 rpm for 10 min in a horizontal centrifuge, the supernatant was filtered using a 0.45 μm filter to obtain the filtered culture medium.

[0106] 3. Purification and incubation: Use 0.15M NaCl and 20mM Na2HPO4 to make 10xBufferA solution, add 10xBuffer to the filtered culture fluid at a ratio of filtered culture fluid: 10xBuffer = 9:1, then add 50μL ProteinA filler to the filtered culture fluid, and incubate at 25℃ for 2h.

[0107] 4. Purification: Assemble the gravity column, wet the filter pad and add 200uL ProteinA filler, dilute 10x Buffer to 1x and pass 100μL through the column to balance the filler, add the incubated sample after equilibration and pass the column twice, then elute 100μL with 1xBuffer, use 4mL 0.1M glycine for elution, and add 440μL Tris NaCl after elution.

[0108] 5. Ultrafiltration: After purification, use a 10k ultrafiltration column for ultrafiltration, and use PBS for ultrafiltration replacement twice to collect the ultrafiltrate.

[0109] Results: The purity of the monoclonal antibody purified by gravity column affinity chromatography was above 99% as detected by SDS-PAGE. Figure 6 , Lane M is a molecular weight marker. Example 7

[0110] Determination of the binding ability of monoclonal antibodies to VZV-gE

[0111] 1. Coating antigen: Dilute VZV-gE protein to 2 μg / mL, spread 100 μL / mL well on a 96-well ELISA plate, and incubate at 4°C overnight.

[0112] 2. Blocking: Wash the plate three times with PBST solution, add 100 μL of 5% BSA solution to each well, and block at 37°C for 1.5 h.

[0113] 3. Sample incubation: Wash the plate 5 times with PBST solution, dilute the 5 candidate antibodies to 10 μg / mL respectively, and dilute them 4 times. Then add 100 μL of diluted samples to each well and block at 37°C for 1 hour.

[0114] 4. Secondary antibody incubation: Wash the plate 5 times with PBST buffer, dilute the secondary antibody to 2.5 μg / mL, add 100 μL of the diluted secondary antibody to each well, and block at 37°C for 1 hour.

[0115] 5. Color development: Wash the plate 5 times with PBST solution, add 100 μL of TMB color development solution to each well, develop color for 15 minutes at room temperature away from light, then add 50 μL of ELISA stop solution to each well to stop color development.

[0116] 6. Reading: Detect the OD value of each well at 450-630nm on a microplate reader, draw the curve using the Logistic four-parameter fitting method, and calculate the EC50 value of the antibody.

[0117] Results: The binding activity of the five candidate antibodies was Figure 6 As shown, the EC50 value of the monoclonal antibody VD460-1 is 147 ng / mL, which shows that the antibody has good binding activity. Figure 7 . Example 8

[0118] Affinity determination of monoclonal antibodies to VZV-gE

[0119] 1. Antigen labeling: After diluting VZV-gE protein to 200 nM, mix the diluted antigen and diluted fluorescence at a ratio of 1:1, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 4°C, 15,000xg, 10 minutes, and aspirate the supernatant into a new tube.

[0120] 2. Preparation of detection antibody: After calculating the molar concentration of the detection antibody, input it into the detection software. According to the software prompts, dilute the VD460-1 antibody to the first concentration, and then dilute it 2-fold with PBST, for a total of 16 tubes. Dilute the 20nM labeled antigen according to the machine prompts, and then add the diluted antibody at a ratio of 1:1, and incubate in the dark for 10-15 minutes.

[0121] 3. Detection: Use a capillary tube to absorb and place the sample into tubes 1-16 in descending order of concentration. Set the detection temperature to 25°C, test on the machine, and read the value.

[0122] Results: The Kd value of the monoclonal antibody VD460-1 is 13.16 nM, which shows that the antibody has good affinity. Figure 8 . Example 9

[0123] Indirect ELISA antibody detection efficacy

[0124] 1. Determination of minimum detection limit and detection range of antibodies:

[0125] 1.1 Coating antigen: VZV-gE protein and recombinant VZV-gE trimer protein were diluted 2-fold from 1000 ng / mL to 11 wells, and 100 μL / mL was plated on a 96-well ELISA plate at 4°C overnight. PBS was used as a negative control.

[0126] 1.2 Blocking: Wash the plate three times with PBST solution, add 100 μL of 5% BSA solution to each well and block at 37°C for 1 h.

[0127] 1.3 Detection antibody incubation: Wash the plate 5 times with PBST solution, dilute VD460-1 to 1 μg / mL, add 100 μL of the diluted detection antibody to each well, and block at 37°C for 1 h.

[0128] 1.4 Secondary antibody incubation: Wash the plate 5 times with PBST solution, dilute anti-human IgG-HRP at 1:10000 and add 100 μL of diluted anti-human IgG-HRP to each well, block at 37°C for 1 h, and use 1% BSA solution as a negative control.

[0129] 1.5 Color development: Wash the plate 5 times with PBST solution, add 100 μL TMB color development solution to each well, develop color for 15 minutes at room temperature away from light, then add 50 μL ELISA stop solution to each well to stop color development.

[0130] 1.6 Reading: Detect the OD value of each well at 450nm on an ELISA reader.

[0131] 2. Antibody precision determination:

[0132] 2.1 Intra-batch precision: The coated VZV-gE protein and recombinant VZV-gE trimer protein from the same batch were diluted to high, medium and low concentrations, and three replicate wells were set for testing. The mean (X), standard deviation (SD) and coefficient of variation (CV) of the antigen concentration within the test were calculated.

[0133] 2.2 Inter-batch precision: Different batches of coated VZV-gE protein and recombinant VZV-gE trimer protein were diluted to high, medium and low concentrations, respectively. Three replicate wells were set for testing, and the mean (X), standard deviation (SD) and coefficient of variation (CV) of the antigen concentration within the test were calculated.

[0134] Results: The minimum detection limit of VD460-1 for monomeric VZV-gE protein was 15.63 ng / mL, and the minimum detection limit for recombinant VZV-gE trimer protein was 1.95 ng / mL. The specific values ​​are shown in Table 1. The linear range of VD460-1 for monomeric VZV-gE protein was from 1000 mg / mL to 62.5 ng / mL, y=1.160x-1.890, R 2 >0.99See details Fig. 9 ; The linear range of VD460-1 for recombinant VZV-gE trimer protein is from 1000mg / mL to 62.5ng / mL, y=1.718x-1.660, R 2 >0.99See details Fig.10 The intra-batch coefficient of variation of the indirect ELISA method VD460-1 for the detection of monomeric VZV-gE protein was 1.52 to 2.83, and the inter-batch coefficient of variation was 1.02 to 1.98; the intra-batch coefficient of variation of the indirect ELISA method VD460-1 for the detection of recombinant VZV-gE trimer protein was 1.26 to 2.97, and the inter-batch coefficient of variation was 2.12 to 3.22, as shown in Table 2.

[0135] Table 1 Minimum detection limit verification results

[0136]

[0137] Table 2 Intra-test precision verification

[0138]

[0139] Table 3 Inter-experimental precision verification

[0140]

[0141] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibody prepared from a recombinant VZV-gE trimer protein, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are shown in the amino acid sequences of positions 26-33, 51-58 and 97-107 of SEQ ID NO.1, respectively; The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are shown in the amino acid sequences at positions 27-36, 54-56 and 93-100 of SEQ ID NO.2, respectively; The amino acid sequence of the recombinant VZV-gE trimer protein is shown in SEQ ID NO.

5.

2. The antibody encoding gene according to claim 1, characterized in that It comprises the VD460-1H nucleotide sequence shown in SEQ ID NO.3 and the VD460-1L nucleotide sequence shown in SEQ ID NO.

4.

3. An antigen-binding fragment that binds to a VZV-gE antigen protein, characterized in that: Contains VH and VL; The VH comprises amino acids 1 to 118 of the amino acid sequence shown in SEQ ID NO.1; The VL comprises amino acids 1 to 110 having an amino acid sequence as shown in SEQ ID NO.

2.

4. Use of the antibody according to claim 1, the encoding gene according to claim 2 or the antigen-binding fragment according to claim 3 in the preparation of a reagent or a kit for detecting VZV-gE protein.

5. Use of the antibody according to claim 1, the encoding gene according to claim 2 or the antigen-binding fragment according to claim 3 in the preparation of a reagent or a kit for detecting herpes zoster virus.

6. Use of the antibody of claim 1, the encoding gene of claim 2 or the antigen-binding fragment of claim 3 in the preparation of a VZV vaccine evaluation product.

7. A reagent or kit for detecting herpes zoster virus, characterized in that: Containing the antibody according to claim 1 or the antigen-binding fragment according to claim 3.

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