A monoclonal antibody for inhibiting the damage of Zika virus to blood-fetal / blood-testis barrier and its application

Abstract

The present invention relates to the field of monoclonal antibodies and pharmaceutical technology, and discloses a monoclonal antibody for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier and its application. The monoclonal antibody is directed against ITGB4 antigen, and the amino acid sequence includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes a heavy chain CDR1 as shown in SEQ ID NO. 3, a heavy chain CDR2 as shown in SEQ ID NO. 4, and a heavy chain CDR3 as shown in SEQ ID NO. 5; the light chain variable region includes a light chain CDR1 as shown in SEQ ID NO. 6, a light chain CDR2 as shown in SEQ ID NO. 7, and a light chain CDR3 as shown in SEQ ID NO. 8. The monoclonal antibody 7C3 of this scheme can bind to the ZIKV host cell receptor ITGB4, effectively inhibit the infection of ZIKV virus to host cells, and increase the expression of Ifnar1 ‑ / ‑ The survival rate of mouse A129 infected with ZIKV. And the 7C3 antibody in this scheme can effectively inhibit the damage of ZIKV virus infection to pregnant mouse embryos or male mouse testicles.

Description

Technical Field

[0001] The present invention relates to the field of monoclonal antibodies and medical technology, and in particular to a monoclonal antibody capable of inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier and application thereof. Background Art

[0002] Zika virus (ZIKV) belongs to the genus Flavivirus of the family Flaviviridae. Its genome is a single-stranded positive-strand RNA. It is an arbovirus mainly transmitted by Aedes mosquitoes, and its natural host is still unknown. The incubation period of ZIKV infection is still unclear, but it is generally 3-12 days. About 80% of ZIKV-infected people are latently infected, and only about 20% will show mild symptoms. The mechanism by which Zika virus destroys the blood-fetal barrier is extremely complex and specific. Studies have shown that the virus first enters the cell by specifically binding to receptors on the surface of trophoblast cells. After the virus enters the cell, it can not only replicate in the trophoblast cells, but also trigger a series of inflammatory responses, leading to a cytokine storm, and then destroy the expression and distribution of cell junction proteins (such as tight junction proteins, adherens junction proteins, etc.). This destruction impairs the integrity of the blood-fetal barrier, forming a "loophole" and providing more routes for the virus to cross. More seriously, Zika virus infection activates matrix metalloproteinases in placental tissue, which further degrade the extracellular matrix and expand barrier damage. Once the virus breaks through the blood-fetal barrier, it can directly enter the fetal circulatory system, and is particularly likely to invade neural precursor cells, leading to abnormal fetal development and ultimately causing serious consequences such as microcephaly.

[0003] Zika virus also shows its unique invasiveness in its destruction of the blood-testis barrier. Zika virus first triggers a local inflammatory response by infecting macrophages and Leydig cells in the testicular interstitium. This inflammatory response leads to the release of multiple inflammatory factors, which damage the expression of tight junction proteins between supporting cells. At the same time, viral infection also leads to increased levels of reactive oxygen species (ROS), further damaging the integrity of the blood-testis barrier. Once the blood-testis barrier is broken, Zika virus can directly infect spermatogonia and supporting cells, which not only affects the production and maturation of sperm, but also may lead to the long-term presence of the virus in semen. Studies have found that even after the symptoms of acute infection have subsided, viral RNA can still be detected in semen for up to 8 months, and this continued presence increases the risk of sexual transmission. More importantly, viral infection may cause chronic inflammation of testicular tissue, affect the production of testosterone, and ultimately may lead to male reproductive dysfunction. Summary of the invention

[0004] The present invention aims to provide a monoclonal antibody and its application for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier. Based on the host cell receptor ITGB4 of ZIKV, the monoclonal antibody 7C3 with the ability to inhibit the damage of ZIKV to the blood-fetal / blood-testis barrier is screened and obtained, so as to solve the technical problem of the existing Zika virus invasion and damage to the blood-fetal / blood-testis barrier.

[0005] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme: a monoclonal antibody for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier, which is a monoclonal antibody against the ITGB4 antigen, and the amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes: a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 3, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 4, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 5; the light chain variable region includes: a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 7, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 8.

[0006] Preferably, as an improvement, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO.2.

[0007] Preferably, as an improvement, the present solution also provides a nucleic acid molecule, including a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0008] Preferably, as an improvement, the present scheme also provides a composition for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier, with the above-mentioned monoclonal antibody as an active ingredient.

[0009] Preferably, as an improvement, the present invention also provides a composition for preventing or treating diseases caused by Zika virus infection, comprising the above-mentioned monoclonal antibody as an active ingredient.

[0010] Preferably, as an improvement, a pharmaceutically acceptable auxiliary agent is also included.

[0011] Preferably, as an improvement, the present solution also provides an expression cassette comprising the above-mentioned nucleic acid molecule.

[0012] Preferably, as an improvement, the present solution also provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0013] Preferably, as an improvement, the present solution further provides an expression system, comprising a host cell, wherein the host cell comprises any one of the above-mentioned monoclonal antibodies, nucleic acid molecules, expression cassettes or expression vectors.

[0014] Preferably, as an improvement, the present scheme also provides an application of a monoclonal antibody for use in the preparation of a drug for inhibiting Zika virus damage to the blood-fetal / blood-testis barrier or preventing Zika virus infection.

[0015] Technical effects of this solution:

[0016] 1. The present invention uses the ZIKV receptor ITGB4 as an antigen to immunize BALB / c mice, screens monoclonal antibodies through hybridoma technology, detects the neutralizing activity of antibodies using a mouse neutralization experiment, selects antibodies with high neutralizing activity for further evaluation of protective dose, window period and preventive effect, amplifies the variable regions of the antibody light and heavy chains, and analyzes their CDR sequences.

[0017] 2. The mouse monoclonal antibody 7C3 obtained in this protocol can bind to the ZIKV host cell receptor ITGB4, effectively inhibit ZIKV virus infection of host cells, and greatly increase Ifnar1 - / - The survival rate of mouse A129 infected with ZIKV was reduced, thereby effectively reducing the infection rate of ZIKV. In addition, experiments have shown that the 7C3 antibody of this scheme can effectively inhibit the damage of ZIKV virus infection to pregnant mouse embryos, and can also effectively inhibit the damage of ZIKV virus infection to the testicles of male mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the affinity curve between the 7C3 antibody and ITGB4 detected by OCTET experiment in an embodiment of the present invention.

[0019] Figure 2 This is a diagram showing the effect of 7C3 antibody on ZIKV replication in cells detected by flow cytometry in an example of the present invention.

[0020] Figure 3 This is a comparison chart of the results of animal experiments in the examples of the present invention to detect the effect of 7C3 antibody on the survival rate of A129 mice.

[0021] Figure 4 This is a comparison chart of the results of the anatomical experiment in the examples of the present invention to examine the effects of 7C3 antibodies on the development of the placenta and embryos of pregnant mice.

[0022] Figure 5 This is a comparison chart of the results of the anatomical experiment in the examples of the present invention to examine the effect of 7C3 antibody on the ZIKV viral load in the testicles of male mice.

[0023] Figure 6 This is a comparison chart of the results of the anatomical experiment examining the effect of 7C3 antibody on testicular damage in male mice in the examples of the present invention.

[0024] Figure 7This is a comparison chart of the results of tissue staining sections of the anatomical experiment to examine the effect of 7C3 antibody on testicular damage in male mice in the examples of the present invention. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0026] Program Overview

[0027] This solution provides a monoclonal antibody for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier, which is a monoclonal antibody against ITGB4 antigen, and the amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes: a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 3, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 4, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 5; the light chain variable region includes: a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 7, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 8. The amino acid sequence of the heavy chain variable region 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.

[0028] The present invention also provides a composition for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier, which contains the above-mentioned monoclonal antibody as an active ingredient.

[0029] The present invention also provides a composition for preventing or treating diseases caused by Zika virus infection, which contains the above-mentioned monoclonal antibody as an active ingredient.

[0030] The above two compositions further include pharmaceutically acceptable adjuvants.

[0031] The present embodiment also provides a nucleic acid molecule, including a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0032] This solution also provides an expression cassette or expression vector, comprising the above-mentioned nucleic acid molecule.

[0033] The present solution also provides an expression system, comprising a host cell, wherein the host cell comprises any one of the above-mentioned monoclonal antibodies, nucleic acid molecules, expression cassettes or expression vectors.

[0034] The present scheme also provides an application of a monoclonal antibody, which is used in the preparation of a drug for inhibiting Zika virus damage to the blood-fetal / blood-testis barrier or preventing Zika virus infection.

[0035] Materials used in the examples

[0036] 1. Experimental animals, cells, and strains

[0037] Female BALB / c mice (6-8 weeks) (Viton River), male Kunming mice (6-8 weeks) (Viton River), Ifnar1 - / - Mouse A129 (purchased from Jackson Lab by Vitallife), SP2 / 0 cells (preserved in the laboratory).

[0038] 2. Experimental reagents and consumables

[0039] The materials and reagents required for this experiment are as follows: 96-well ELISA plate (NEST); cell culture consumables including T25 culture flasks, cryovials, 20 mL and 50 mL sterile centrifuge tubes (all Corning), and 6-, 24-, and 96-well cell culture plates (COSTAR); PVDF membrane (Millipore); culture medium and additives including 1640 culture medium, double antibody, serum (all Gibico) and DMEM culture medium (Kccell); kits including cell / bacterial total RNA extraction kit, plasmid extraction kit (all Tiangen), RevertAid First Strand cDNA synthesis kit (Thermo), Easysee Western Blot Blot kit (Quanshijin), mouse monoclonal antibody Ig class / subclass identification reagent (Biolong); chromatography and immune-related materials include Protein-G affinity chromatography column (GE), HRP-labeled goat anti-mouse IgG (Thermo), complete and incomplete Freund's adjuvant (both Sigma); other reagents include TMB chromogenic substrate (Solabo), PEG (Sigma), BoNTA toxicity standard (our laboratory), SDS-PAGE sample buffer (Sangon), and DMSO (Inokai).

[0040] 3. Solution preparation

[0041] (1) Preparation of experimental buffer. Coating buffer (pH 9.6): Na2CO3 1.59 g and NaHCO3 2.93 g dissolved in 1 L ddH2O. PBS (pH 7.4): NaCl 8 g, KCl 0.2 g, Na2HPO4·12H2O 3.58 g, KH2PO4 0.24 g dissolved in 1 L ddH2O. PBST: PBS with 0.05% Tween 20. Protein-G purification binding buffer (pH 7.0): NaH2PO4 1.47 g, Na2HPO4·12H2O 1.1 g dissolved in 1 L ddH2O; elution buffer (pH 2.7): glycine 7.507 g / L. 1 M Tris-HCl (pH 7.9): Tris-Base 12.11 g dissolved in 100 mL ddH2O. Transfer buffer (1×): 2.9 g glycine, 5.8 g Tris-Base, 0.37 g SDS, 200 mL methanol, dilute to 1 L.

[0042] (2) Preparation of culture medium. 1640 and DMEM complete culture medium: Add 10% Gibico or Sigma serum and 1% double antibody to the basal culture medium. HAT selection medium: Dilute 50×HAT to 1× in DMEM containing 20% ​​Sigma serum. LB liquid culture medium: 10 g NaCl, 5 g yeast extract, 10 g peptone, dissolved in 1 L ddH2O. LB solid culture medium: Add 1.5% agar to LB liquid culture medium.

[0043] (3) Preparation of other solutions. Blocking solution: 5% skim milk powder dissolved in PBST. 2% BSA solution: 2 g BSA dissolved in 100 mL ddH2O. Cell freezing solution: 90% Sigma serum mixed with 10% DMSO. All solutions must be prepared using analytical grade reagents and sterilized or filtered according to experimental requirements. Storage conditions and expiration dates should be strictly followed according to experimental requirements.

[0044] Methods used in the examples

[0045] 1. The preparation and purification method of monoclonal antibody 7C3 comprises the following steps:

[0046] (1) According to the monoclonal antibody preparation process, female BALB / c mice aged 6 to 8 weeks were selected. For the first immunization, 50 μg of antigen ITGB4 was mixed with complete Freund's adjuvant and injected subcutaneously at multiple points. On day 21, the same dose of ITGB4 was mixed with incomplete Freund's adjuvant for the second immunization. On day 42, the same dose of ITGB4 was directly injected intraperitoneally without adjuvant to complete the third immunization. On day 56, 500 μg of ITGB4 was injected intraperitoneally to boost immunization.

[0047] (2) Three days after booster immunization, mice were killed by cervical dislocation and disinfected with 75% alcohol. Splenocytes were isolated under sterile conditions and mixed with SP2 / 0 myeloma cells at a ratio of 1:5. Cell fusion was performed by PEG method. Positive clones were screened and cloned by limiting dilution method.

[0048] (3) After obtaining a hybridoma cell line that stably secretes antibodies, its culture supernatant was co-incubated with Zika virus (ZIKV) to measure the inhibitory effect on ZIKV infection of Vero cells, and the monoclonal antibody 7C3 with the best inhibitory effect was screened out.

[0049] (4) Antibodies were prepared by in vivo induction. Each BALB / c mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. Two weeks later, 1×10 6 10 days after injection, the ascites was collected, salted out using saturated ammonium sulfate, and the antibody was purified by Protein-G affinity chromatography.

[0050] 2. OCTET method to determine the affinity of monoclonal antibody 7C3 to ITGB4

[0051] (1) Experimental preparation: Use OCTET biolayer interferometer and Protein A biosensor. Prepare monoclonal antibody 7C3 (10 μg / mL) and ITGB4 protein concentration gradient (200-3.125 nM). All samples and buffers need to be centrifuged to remove impurities.

[0052] (2) Set up reference wells and replicate wells on a 96-well black microplate and arrange the ligand, analyte, and buffer in a reasonable manner. Ensure that the experimental conditions are constant, including temperature (25°C) and shaking speed (1000 rpm).

[0053] (3) Each step lasts 60 seconds and is treated with buffer.

[0054] (4) Use the OCTET system to monitor the binding and dissociation processes in real time and record the changes in optical thickness to ensure a stable baseline, good binding curve quality, and reproducible data.

[0055] (5) Use OCTET data analysis software to perform curve fitting and calculate kinetic parameters, including the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD). Evaluate the goodness of fit, compare the binding curves at different concentrations, confirm the reliability of the kinetic parameters, and finally obtain the affinity data of 7C3 and ITGB4.

[0056] 3. Evaluation of the effect of monoclonal antibody 7C3 in protecting mouse survival rate

[0057] In order to detect whether the antibody has a neutralizing effect and whether it can play an antiviral role, 30 6-week-old interferon receptor-deficient mice were divided into 3 groups and intraperitoneally injected with 7C3 antibody (i.e., 7C3 group), IgG (i.e., IgG group) or sterile water (i.e., Mock group). On the second day, mice in the 7C3 group and IgG group were injected with 10 4 FFU ZIKV-SMGC-1 virus, and the Mock group was injected with the same amount of sterile water. The mice were weighed every day before and after the injection to detect weight changes and record the death of the mice. When the weight of the mice dropped below 75% of the initial weight, the mice were killed and recorded as dead.

[0058] 4. Monoclonal antibody 7C3 inhibits ZIKV infection of sensitive cells

[0059] (1) Divide the cells into 6-well plates and culture them for 12 to 24 hours until the cell confluence is about 70%.

[0060] (2) Take a certain amount of antibody, dilute it with culture medium, add it to the cells, and incubate at 4°C for 60 minutes.

[0061] (3) The treated cells were infected with ZIKV virus at an MOI of 0.5 and incubated at 4°C for 1 hour.

[0062] (4) Aspirate the supernatant and wash three times with cold PBS.

[0063] (5) Cells are directly processed for experimental analysis or cultured for a specific period of time for experimental analysis.

[0064] 5. Virus titer detection

[0065] (1) The Vero cells in a 10 cm dish were digested with trypsin and then resuspended in DMEM containing 10% serum.

[0066] (2) Count the resuspended cells and divide them into 96-well plates, with approximately 1,000 cells per well. Continue culturing for 12 to 24 hours.

[0067] (3) Take 100 μl of the virus stock solution and dilute it in multiples from 10 to 108 using culture medium containing 2% serum.

[0068] (4) Add 100 μl of each dilution into each well of a 96-well plate. Repeat 10 wells for each dilution and set up two wells as negative controls.

[0069] (5) Place the cells in an incubator at 37°C and 5% CO2 for 5 days, and observe the cell cytopathic effects every day.

[0070] (6) Calculate the ratio of the lesions above and below 50%, ratio = (lesion rate above 50% - 50%) / (lesion rate above 50% - lesion rate below 50%).

[0071] (7) lgTCID50 = distance ratio × difference between the logarithms of dilution + the logarithm of the dilution with a lesion rate higher than 50%. TCID50 means: when the virus is diluted and inoculated into 100 μl of cells, 50% of the cells will become lesions.

[0072] 6. Amplification of antibody heavy chain and light chain variable region genes

[0073] Total RNA from cells was extracted using the Tiangen RNAprep pure Cell / Bacteria Kit and reverse transcribed into cDNA using the Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit.

[0074] (1) Recovery and culture of positive clone cells

[0075] Take out the cryopreservation tube containing the cell line from the liquid nitrogen tank and immediately put it into 37℃ water to thaw. Immediately centrifuge at 1000r / min for 3 min after thawing. Remove the supernatant, take 1 mL of DMEM cell culture medium and quickly add it to the cryopreservation tube to resuspend the cells, and transfer it to a cell culture bottle with 10 mL of DMEM cell culture medium. When the cells grow to the proliferation stage, take 1 mL into the cell culture bottle with 10 mL of DMEM cell culture medium, and extract total RNA when the cells grow to the proliferation stage.

[0076] (2) Extraction of total RNA from positive clones

[0077] The cells were gently blown up and counted, and the cells were collected in a centrifuge tube and centrifuged at 1000 r / min for 3 min. The supernatant was removed (the cell amount was controlled to be no more than 5×10 6 Total RNA was extracted according to the instructions of Tiangen Cell / Bacteria Total RNA Extraction Kit (finally eluted with 30 µL of nuclease-free ddH2O).

[0078] (3) Reverse transcription into single-stranded cDNA

[0079] The system was designed according to the instructions of Thermo Scientific RevertAid First Strand cDNA Synthesis Kit as shown in Table 1.

[0080] Table 1 Thermo Scientific RevertAid First Strand cDNA Synthesis Kit Instruction Manual Design System

[0081]

[0082] After the reagents melted, mix them according to the above system and place them on ice. Centrifuge for 3-5 seconds and incubate them at 65°C for 5 minutes.

[0083] Place the test tube in an ice bath and add the components listed in Table 2.

[0084] Table 2 Additive components details

[0085]

[0086] Mix gently and centrifuge for 3-5 seconds, incubate at 42℃ for 60 minutes. Place the reaction system in a 70℃ metal bath for 5 minutes to terminate the reaction. Finally, store the cDNA in a -70℃ refrigerator.

[0087] The cDNA of the antibody was used as a template, and degenerate primers (Li Jing, Wang Yan, Li Quanxi, etc. Cloning of the V region gene of the anti-gastric cancer mouse monoclonal antibody 3H11 and expression of human-mouse chimeric light chain. Chinese Journal of Microbiology and Immunology. Vol. 17, No. 3, May 1997) were used to amplify the variable region genes of the antibody light chain and heavy chain respectively. The variable region gene of the antibody was connected to the pMD-18T plasmid and then transferred to DH5α. The bacteria with positive results of colony PCR identification were sent for sequencing, and the antibody gene sequence was intercepted by comparing the PCR primers with the measured sequence. After checking its reading frame, the measured antibody variable region sequence was analyzed using the database IMGT to obtain its CDR region sequence.

[0088] (4) Target fragment PCR

[0089] The obtained antibody cDNA was used as a template for PCR. The degenerate PCR primers used are shown in Table 3.

[0090] Table 3 Antibody variable region amplification primer sequence list

[0091]

[0092] There are 5 forward primers for antibody light chains and 4 forward primers for antibody heavy chains. The PCR reaction system was designed according to the instruction manual of Trans Taq DNA Polymerase HiFi Fidelity (HiFi) as shown in Table 4.

[0093] Table 4 Trans Taq DNA Polymerase HiFi Fidelity (HiFi) Instructions for designing PCR reaction system

[0094]

[0095] The program used touchdown PCR, with pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing temperature decreased from 65°C to 56°C in the first ten cycles, and maintained at 56°C for the next 20 cycles. Extension at 72°C for 90 s, a total of 30 cycles, final extension at 72°C for 10 min, and storage at 20°C.

[0096] (5) Gel excision and recovery of gene fragments

[0097] PCR products were separated by 1% agarose gel, and the gene fragments of the light chain variable region and the heavy chain variable region were recovered respectively. If there were non-specific bands, the bands between 250-500 bp were selected for recovery.

[0098] (6) Identification of gene segments of light chain variable regions and heavy chain variable regions

[0099] The recovered target fragment was connected to pMD-18T and transferred to DH5α for blue-white spot screening. The white spots were selected for colony PCR identification, and the successfully identified bacterial liquid was sent for sequencing.

[0100] The ligation reaction system is shown in Table 5.

[0101] Table 5 Ligation reaction system

[0102]

[0103] After mixing the system, incubate at 16℃ overnight. The next day, mix the ligation product with 100 µL DH5α competent cells on ice, ice bath for 30 min, heat shock at 42℃ for 90 s, ice bath for 2 min, add 200 µL of non-resistant LB medium, mix well, and activate at 37℃ shaker at 200r / min for 0.5 h. Spread the activated bacteria evenly on AMP-resistant LB solid medium and culture in a 37℃ incubator overnight.

[0104] A single clone was picked and cultured in 5 mL AMP-resistant LB liquid medium for 8 h. The bacterial solution was used as a template for bacterial solution PCR identification, and the PCR reaction system was designed as shown in Table 6.

[0105] Table 6 Design of PCR reaction system

[0106]

[0107] The program used touchdown PCR, with pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing temperature decreased from 65°C to 56°C in the first ten cycles, and maintained at 56°C for the next 20 cycles. Extension at 72°C for 90 s, a total of 30 cycles, final extension at 72°C for 10 min, and storage at 20°C.

[0108] After the PCR products were separated and identified using 1% agarose gel, the bacterial solution with positive PCR results was sent for sequencing. The plasmid was extracted from the bacterial solution with successful sequencing and the plasmid was preserved.

[0109] (7) Use RCR primers to find the antibody variable region sequence from the sequence obtained by sequencing

[0110] Use the software DNAMAN to align the forward primer and reverse primer with the sequence obtained by sequencing, and the middle sequence is the gene sequence of the monoclonal antibody variable region. Save the gene sequence of the monoclonal antibody variable region.

[0111] 7. Evaluation of the efficacy of antibodies in protecting the placenta of pregnant mice against Zika virus infection

[0112] 8-9 week old interferon-deficient female mice were mated with wild-type C57 mice, and 60 E5.5 female mice were selected and then divided into 3 groups. One group was injected with 500 μg of IgG (i.e., IgG group), one group was injected with 500 μg of 7C3 (i.e., 7C3 group), and the last group was injected with the same dose of sterile water (i.e., Mock group). 4 FFU ZIKV-SMGC-1 virus (the Mock group was injected with the same dose of sterile water) was dissected on the 8th day after virus infection, and the placenta size was recorded and weighed. The placenta was placed in 8% paraformaldehyde (PFA), and after 72 hours of immersion, it was replaced with 60% ethanol solution, then embedded in paraffin, sectioned, and stained with hematoxylin-eosin. At the same time, a portion of the placenta from each group of mice was homogenized and the virus titer was tested.

[0113] 8. Determination of viral load in male mouse testis

[0114] Tissues were weighed and homogenized in 200 μl PBS. Samples were centrifuged at 10,000 × g for 10 min and then stored at −80°C. ZIKV RNA was extracted from tissue and serum samples, and ZIKV RNA levels were measured by Taqman one-step qRT-PCR. Viral loads were expressed as viral RNA equivalents per gram or milliliter on a log10 scale after calculation using a standard curve prepared by serial ten-fold dilutions of ZIKV RNA. ZIKV RNA was detected using published primers.

[0115] Example

[0116] 1. Screening, preparation and identification of monoclonal antibodies

[0117] Female BALB / c mice (6-8 weeks) were selected for three ITGB4 antigen immunizations, and the No. 7 mouse with the highest titer (30,000) was selected for booster immunization. The spleen cells of No. 7 mouse and myeloma cells SP2 / 0 were fused by cell fusion technology to screen hybridoma cells, and the positive wells were cloned to finally obtain 30 positive cell lines. The neutralization activity of ZIKV was preliminarily determined by the cell supernatant to select the one with the best activity. After culturing the hybridoma cells, the supernatant was affinity purified by Protein-G. The purified antibodies were quantitatively evaluated for activity, and the one with the best activity was selected for Western blot identification of its specific binding to the antigen ITGB4.

[0118] After two rounds of neutralization activity screening, it was finally determined that antibody No. 7 (7C3) had the highest affinity. OCTET determined the affinity constant K of 7C3 and ITGB4. D =30.9 pM( Figure 1 ).

[0119] 2. 7C3 antibody can effectively inhibit ZIKV virus infection of host cells

[0120] To further verify that the 7C3 antibody can effectively inhibit the infection of ZIKV, a neutralization inhibition experiment was conducted, that is, the host cells were incubated with the 7C3 antibody to block the binding sites of ITGB4 and ZIKV on the cell surface, and then the cells were infected by incubating with ZIKV at 4°C for 1 hour. After culturing at 37°C for a period of time, the replication of ZIKV in the host cells was detected.

[0121] 7C3 (80 μg / ml) (i.e., 7C3 group), rabbit IgG (i.e., IgG group) and sterile water (i.e., Mock group) were incubated with Hun-7 and Vero cells, respectively. Then, the cells in the 7C3 group and IgG group were infected with ZIKV for 1 hour at 4°C (the Mock group was not infected). After 1 hour, they were washed three times with pre-cooled culture medium, added with 10% FBS DMEM and placed in an incubator for 24 hours, and the replication of ZIKV in cells was detected by flow cytometry. The results showed that compared with IgG, 7C3 antibody can effectively inhibit the infection of ZIKV in two sensitive cell lines, Hun-7 and Vero ( Figure 2 ).

[0122] 3. 7C3 antibody improves the survival rate of ZIKV-infected mice

[0123] Five to six-week-old Ifnar1- / - male mice were randomly divided into three groups and injected intramuscularly with 500 μg 7C3 (i.e., 7C3 group), IgG (i.e., IgG group), or sterile water (i.e., Mock group). On the second day, mice in the 7C3 and IgG groups were injected intramuscularly with 10 4 The FFU ZIKV-SMGC-1 and Mock groups were injected with the same amount of sterile water. The health status of the mice was then monitored every day.

[0124] like Figure 3 As shown, after the injection of ZIKV, the mice in the IgG group began to die on the 6th day, and this phenomenon continued until about the 10th day. Only about 20% of the mice did not die and survived. The mice in the 7C3 group began to die on the 8th day, which was 2 days later than the IgG group. The mice in the 7C3 group stopped dying after the 13th day, and about 60% of the mice did not die and survived. The survival rate of mice in the 7C3 group was 60%, which was much higher than the survival rate of mice in the IgG group (20%). The survival rate of mice in the Mock group, that is, mice that were not infected with the virus, was 100%. Therefore, the 7C3 antibody can effectively protect mice from the lethal effect of Zika virus infection ( Figure 3 ).

[0125] 4. 7C3 antibody protects mice against placental damage caused by Zika virus infection

[0126] ZIKV infection in pregnant women can cause microcephaly in the fetus. A retrospective study found that 29% of embryos in pregnant women infected with ZIKV showed developmental abnormalities, including microcephaly, intrauterine growth restriction, fetal death, and stillbirth. In the process of fetal development, the first three months of pregnancy is a critical period for fetal brain development.

[0127] To investigate the relationship between microcephaly and ZIKV infection in pregnant women, a study reported a mouse infection model during pregnancy. ZIKV infection can effectively overcome the immune response mediated by type I interferon receptor (IFNAR1) in humans and primates, but not in mice. Therefore, the researchers used a double-copy deletion of Ifnar1 to - / - The mother mice mated with wild-type male mice, and the resulting embryonic pups acquired an Ifnar1 gene, thereby obtaining a complete infectious agent response. Antibodies were injected on day 5.5 of pregnancy, and on day 6.5 (the gestation period of mice is between 19-21 days, and day 6.5 is equivalent to 3 months of human pregnancy, which is a critical period for fetal brain development) the ZIKV virus was injected into the soles of the feet to simulate mosquito bite infection. The embryos were processed on days 13.5 and 16.5 to detect the characteristics of the placenta and fetus.

[0128] Based on the above model, 36 mice were divided into Mock group, IgG group and 7C3 group. On the 5.5th day of pregnancy, 500 μg of antibody was injected into each group (specifically, 500 μg of 7C3 was injected into the 7C3 group, the same amount of IgG was injected into the IgG group, and the same amount of sterile water was injected into the Mock group). On the 6.5th day, 10 4 FFU ZIKV-SZ01 virus (Mock group injected with the same amount of sterile water), mice were killed on the 8th day of virus infection for testing. The results showed that the embryos in the IgG group were significantly shrunken and smaller (smaller than the embryos in the 7C3 group), indicating that the 7C3 antibody effectively protected the mouse placenta and embryos from ZIKV damage ( Figure 4 ).

[0129] 5. 7C3 antibody protects male mice against testicular damage caused by Zika virus infection

[0130] To confirm the preventive and protective effects of 7C3 on the blood-testis barrier, the ZIKV infection in the testes of male mice was evaluated on day 21. The results showed that the viral load in the testes of male mice injected with 7C3 antibody in advance (i.e., 7C3 group) was relatively low. In contrast, the viral load in the testes of male mice injected with monoclonal antibody IgG (isotype control IgG group) in advance was significantly increased ( Figure 5 ).

[0131] In addition, 7C3 antibodies can protect the normal growth and development of the testicles from the effects of ZIKV. Compared with the pre-injection of IgG, the testicles of male mice were more able to grow and develop normally after the pre-injection of 7C3 ( Figure 6 Finally, by staining testicular tissue cell apoptosis reagents, it was found that 7C3 monoclonal antibody can effectively protect the integrity of the testicles of male mice ( Figure 7 ).

[0132] 6. Amplification and analysis of antibody heavy chain and light chain variable region sequences

[0133] Total RNA from cells was extracted using the Tiangen RNAprep pure Cell / Bacteria Kit RNAprep pure cultured cell / bacteria total RNA extraction kit, and reverse transcribed into cDNA using the Thermo Scientific RevertAid First StrandcDNA Synthesis Kit. Using the obtained antibody cDNA as a template, degenerate primers were used to amplify the variable region genes of the antibody light chain and heavy chain, respectively. The antibody variable region genes were connected to the pMD-18T plasmid and then transferred to DH5ɑ. The bacteria with positive results from colony PCR were sent for sequencing, and the sequences of the variable regions of the light and heavy chains of the 7C3 antibody and their CDR sequences were measured as shown in Table 7.

[0134] Table 77C3 antibody light and heavy chain variable region sequences and CDR sequences

[0135]

[0136] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A monoclonal antibody that inhibits Zika virus damage to the blood-fetal / blood-testis barrier, characterized in that: It is a monoclonal antibody against ITGB4 antigen, the amino acid sequence of the monoclonal antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes: a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 3, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 4, and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO. 5; the light chain variable region includes: a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO. 6, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO. 7, and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO.

8.

2. A monoclonal antibody for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region 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.

3. A composition for inhibiting the damage of Zika virus to the blood-fetal / blood-testis barrier, characterized in that: The monoclonal antibody according to claim 1 or 2 is used as an active ingredient.

4. A composition for preventing or treating diseases caused by Zika virus infection, characterized in that: The monoclonal antibody according to claim 1 or 2 is used as an active ingredient.

5. A composition for preventing or treating diseases caused by Zika virus infection according to claim 4, characterized in that: Pharmaceutically acceptable adjuvants are also included.

6. A nucleic acid molecule, characterized in that: Comprising a nucleic acid molecule encoding the monoclonal antibody of claim 3.

7. An expression cassette, characterized in that: Comprising the nucleic acid molecule of claim 6.

8. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 6.

9. An expression system, characterized in that: The invention comprises a host cell, wherein the host cell comprises any one of the monoclonal antibody according to claim 1 or 2, the nucleic acid molecule according to claim 6, the expression cassette according to claim 7 or the expression vector according to claim 8.

10. A use of a monoclonal antibody, characterized in that: The monoclonal antibody according to claim 1 or 2 is used in the preparation of a drug for inhibiting Zika virus damage to the blood-fetal / blood-testis barrier or preventing Zika virus infection.

Images