CN109897104B - Human adenovirus type 7 monoclonal antibody 3-3E and its application - Google Patents

Abstract

The invention discloses a human adenovirus 7 monoclonal antibody 3-3E and application thereof. The invention provides a monoclonal antibody, which comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions HCDR1, HCDR2 and HCDR 3; the light chain variable region comprises three complementarity determining regions, LCDR1, LCDR2, and LCDR 3; the HCDR1, the HCDR2 and the HCDR3 are sequentially shown as 26 th to 33 th, 51 th to 58 th and 97 th to 110 th from the N end of a sequence 2in a sequence table; the LCDR1, the LCDR2 and the LCDR3 are sequentially shown as 27 th to 32 th, 50 th to 52 th and 89 th to 96 th from the N end of a sequence 4 of a sequence table. Based on clinical requirements, the inventor discovers the anti-human adenovirus 7 antibody 3-3E, which is used for preventing and treating adenovirus infection and has important biological and medical significance.

Description

Human adenovirus type 7 monoclonal antibody 3-3E and its application

technical field

The present invention relates to a human type 7 adenovirus monoclonal antibody 3-3E and its application.

Background technique

Human adenovirus belongs to the Adenoviridae family of mammalian adenovirus. Human adenovirus was first discovered in 1953. It was isolated and cultured from the atrophied tonsil-like tissue of healthy people; the genome of the virus is double-stranded DNA with a total length of about 36kb. DNA and viral structural proteins combine to form the virus core. The virus has no envelope, and the core is coated with a capsid. The capsid consists of 252 capsomers, of which 240 are hexon proteins, 12 penton proteins, and the outer shell. It has a regular icosahedral structure with a diameter of about 80-110 nm.

Adenoviruses that have been found so far have a total of 7 subgroups A-G, 67 different serotypes, of which 55 subtypes can infect humans and cause disease. Adenoviruses most commonly infect the respiratory tract and cause respiratory system diseases. In addition, some viruses can also cause urinary and gastrointestinal infections. Studies have shown that adenoviruses that cause respiratory tract infections mainly include groups A, C, E and B1, among which Mainly B1 subgroup, B2 subgroup can infect human urinary system, F and D groups can infect human gastrointestinal system and conjunctival system respectively. Currently known adenoviruses that cause respiratory infections in the world include B1 subgroup types 1, 2, 3, 4, 7, 14 and 55, of which types 3, 4, 7 and 14 are the most common types that cause outbreaks The type of adenovirus has caused outbreaks of respiratory adenovirus in Jiangsu and Taiwan Provinces of my country, as well as South Korea, Singapore, and Malaysia. Since 2008, there have been many outbreaks of infectious diseases spread through the respiratory tract in different regions of my country. The epidemic spreads widely and is highly contagious. The laboratory pathogen detection identified as group B adenovirus type 7, type 55 and type 14 respectively. .

Under normal circumstances, when a virus infects the human body, the human immune system can stimulate the humoral and cellular immune responses and gradually control the infection and clear the virus. Most viruses infect the human body about 3 days after the onset of the disease, and the patient's body begins to produce IgM and IgG. Later, most of them began to produce in 7-10d, and then gradually increased, generally reaching a peak in about 1 month. Therefore, neutralizing antibodies have always played an important role in the recovery of virus infection. Adenovirus infection in the human body can also induce a strong humoral immune response and produce specific antibodies. Studies have shown that the body can produce effective immunity against re-infection with the same type of adenovirus, and can produce long-term immune protection (up to more than 10 years). Induce neutralizing antibodies produced by the body. According to research, 40% to 60% of people aged 6 to 15 in my country have type 1, 2 and 5 neutralizing antibodies, and the mother's antibodies can protect the baby from severe adenovirus infection. Therefore, adenovirus neutralizing antibody is considered to be an effective and specific anti-adenovirus treatment method, and the development of adenovirus neutralizing antibody should be a major development direction for the development of adenovirus infection treatment drugs in the future.

At present, there is no specific therapeutic drug for adenovirus in the world, and drug development is mostly limited to chemical drugs—nucleoside analogs against DNA viruses, while the research on specific biological drugs for adenovirus is still blank.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a human type 7 adenovirus monoclonal antibody 3-3E and its application.

The present invention first provides a monoclonal antibody comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementarity determining regions HCDR1, HCDR2 and HCDR3; the light chain variable region Including three complementary determining regions LCDR1, LCDR2 and LCDR3;

The amino acid sequence of the HCDR1 is as follows (a1) or (a2) or (a3):

(a1) the amino acid sequence shown in the 26th-33rd position from the N-terminal of Sequence 2 of the sequence listing;

(a2) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown in the 26th to 33rd position of the N-terminal in Sequence 2 of the sequence listing;

(a3) an amino acid sequence with 75% or more homology and the same function as the amino acid sequence shown in the 26th to 33rd position from the N-terminus of Sequence 2 of the Sequence Listing;

The HCDR2 is (a4) or (a5) or (a6) as follows:

(a4) the amino acid sequence shown in the 51st-58th position from the N-terminal of Sequence 2 of the Sequence Listing;

(a5) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown at positions 51-58 of the N-terminal in Sequence 2 of the Sequence Listing;

(a6) an amino acid sequence with 75% or more homology and the same function as the amino acid sequence shown in the 51-58th position from the N-terminal of Sequence 2 of the Sequence Listing;

The HCDR3 is (a7) or (a8) or (a9) as follows:

(a7) the amino acid sequence shown in the 97th-110th position from the N-terminal of Sequence 2 of the Sequence Listing;

(a8) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown in the 97th-110th position of the N-terminal in Sequence 2 of the sequence listing;

(a9) an amino acid sequence with 75% or more homology and the same function as the amino acid sequence shown in the 97th to 110th position from the N-terminus of Sequence 2 of the Sequence Listing;

The amino acid sequence of LCDR1 is as follows (b1) or (b2) or (b3):

(b1) the amino acid sequence shown in the 27th-32nd position from the N-terminal of Sequence 4 of the sequence listing;

(b2) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown at positions 27-32 of the N-terminal in Sequence 4 of the sequence listing;

(b3) an amino acid sequence with 75% or more homology and the same function as the amino acid sequence shown in the 27th-32nd position from the N-terminus of Sequence 4 of the Sequence Listing;

The amino acid sequence of LCDR2 is as follows (b4) or (b5) or (b6):

(b4) the amino acid sequence shown in the 50th-52nd position from the N-terminal of Sequence 4 of the sequence listing;

(b5) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown at positions 50-52 of the N-terminal in Sequence 4 of the Sequence Listing;

(b6) an amino acid sequence with 75% or more homology and the same function as the amino acid sequence shown in the 50th to 52nd position from the N-terminus of Sequence 4 of the Sequence Listing;

The amino acid sequence of LCDR3 is as follows (b7) or (b8) or (b9):

(b7) the amino acid sequence shown in the 89th-96th position from the N-terminal of Sequence 4 of the sequence listing;

(b8) an amino acid sequence with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues from the amino acid sequence shown at positions 89-96 of the N-terminal in Sequence 4 of the Sequence Listing;

(b9) An amino acid sequence having 75% or more homology with the amino acid sequence shown from the 89th to 96th position from the N-terminal of SEQ ID NO: 4 of the Sequence Listing and having the same function.

The amino acid sequence of the heavy chain variable region is as follows (c1) or (c2) or (c3):

(c1) the amino acid sequence shown in Sequence 2 of the Sequence Listing;

(c2) an amino acid sequence with the same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues to the amino acid sequence shown in Sequence 2 of the Sequence Listing;

(c3) an amino acid sequence that has 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 of the Sequence Listing and has the same function;

The amino acid sequence of the light chain variable region is as follows (c4) or (c5) or (c6):

(c4) the amino acid sequence shown in Sequence 4 of the Sequence Listing;

(c5) an amino acid sequence with the same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues to the amino acid sequence shown in Sequence 4 of the sequence listing;

(c6) An amino acid sequence having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 4 of the Sequence Listing and having the same function.

The present invention also protects the gene encoding the antibody.

The gene encoding the heavy chain variable region of the antibody is as follows (d1) or (d2) or (d3):

(d1) the DNA molecule shown in Sequence 1 of the Sequence Listing;

(d2) a DNA molecule having 75% or more homology with the nucleotide sequence defined in (d1) and encoding the heavy chain variable region described in claim 1 or 2;

(d3) a DNA molecule that hybridizes to the nucleotide sequence defined in (d1) or (d2) under stringent conditions and encodes the heavy chain variable region of claim 1 or 2;

The gene encoding the light chain variable region of the antibody is as follows (d4) or (d5) or (d6):

(d4) the DNA molecule shown in Sequence 3 of the Sequence Listing;

(d5) a DNA molecule having 75% or more homology with the nucleotide sequence defined in (d4) and encoding the heavy chain variable region described in claim 1 or 2;

(d6) A DNA molecule that hybridizes to the nucleotide sequence defined in (d4) or (d5) under stringent conditions and encodes the heavy chain variable region of claim 1 or 2.

The present invention also protects the application of any of the above monoclonal antibodies in the preparation of a medicament for preventing and/or treating diseases caused by adenovirus infection.

The present invention also protects the application of any of the monoclonal antibodies described above in the preparation of products; the uses of the products are as follows (e1) and/or (e2):

(e1) inhibiting adenovirus;

(e2) Neutralization of adenovirus.

The present invention also protects a medicament for preventing and/or treating diseases caused by adenovirus infection, the active ingredient of which is any of the monoclonal antibodies described above.

The present invention also protects a product whose active ingredient is any of the monoclonal antibodies described above; the uses of the product are as follows (e1) and/or (e2):

(e1) inhibiting adenovirus;

(e2) Neutralization of adenovirus.

Any of the above-mentioned adenoviruses are human adenoviruses. The human adenovirus is human adenovirus type 7.

Based on clinical needs, the inventors of the present invention have discovered the anti-human adenovirus type 7 antibody 3-3E, which is used for the prevention and treatment of adenovirus infection, and has important biological and medical significance.

Description of drawings

Figure 1 shows the expression and purification of 3-3E.

Figure 2 shows the detection of HAdV7 by SDS-PAGE electrophoresis.

Figure 3 shows the electron microscope detection of HAdV7.

Figure 4 is an antigen specificity analysis of 3-3E.

Figure 5 is an affinity analysis of 3-3E.

Figure 6 shows the construction of an animal model of adenovirus infection.

Figure 7 is an analysis of the effect of 3-3E vaccination on the body weight of CB-17SCID mice.

Figure 8 shows the protective effect of 3-3E on HAdV7-infected CB-17SCID mice.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

HAdV7 virus: human/CHN/GZ6965/2001, described in the literature: Yu Z, Zeng Z, Zhang J, et al. Fatal Community-acquired Pneumonia in Children Caused by Re-emergent HumanAdenovirus 7d Associated with Higher Severity of Illness and Fatality Rate[ J]. Scientific Reports, 2016, 6: 37216.; Publicly available from the Academy of Military Medicine of the Chinese Academy of Military Sciences.

Influenza virus antigen: A/swine/Colorado/1/77(H3N2), described in the literature: Karasin AI, Schutten M M, Cooper LA, et al. Genetic characterization of H3N2influenza virusesisolated from pigs in North America,1977-1999:Evidence for wholly human andreassortant virus genotypes[J]. Virus Research, 2000, 68(1): 71-85.; The public can obtain from the Academy of Military Medicine of the Academy of Military Sciences of the Chinese People's Liberation Army.

Example 1. Discovery of Antibodies

The peripheral blood of patients with DENV-1 infection in the convalescent period was collected, and the peripheral blood mononuclear lymphocytes were isolated and collected. The collected peripheral blood mononuclear lymphocytes were added with anti-cell surface corresponding molecular marker antibodies (BD, #555332, #555415, #555441, #560677, #555622) or control antibodies (BD, #555748, #555742, #555751, # 555749, #557872) were labeled, followed by flow sorting, and sorted cells were used for antibody gene amplification. Using the isolated single B cell as a template, amplify the antibody gene, sequence and express the positive clone, and use the purified sample of adenovirus type 7 as the antigen. After a large number of screening, analysis and verification, an antibody sequence was obtained, named as 3-3E antibody.

The amino acid sequence of the heavy chain variable region of the 3-3E antibody is shown in Sequence 2 of the Sequence Listing (wherein, the amino acid residues at positions 26-33 from the N-terminal form CDR1, the amino acid residues at positions 51-58 form CDR2, and the amino acid residues at positions 51-58 form CDR2. Amino acid residues 97-110 constitute CDR3), and the encoding gene is shown in Sequence 1 of the sequence listing.

The amino acid sequence of the light chain variable region of the 3-3E antibody is shown in Sequence 4 of the Sequence Listing (wherein, the 27th-32nd amino acid residues from the N-terminal form CDR1, the 50th-52nd amino acid residues form CDR2, and the 50th-52nd amino acid residues form CDR2. The 89-96 amino acid residues constitute CDR3), and its coding gene is shown in sequence 3 of the sequence table.

The preparation of embodiment 2, 3-3E antibody

1. Construction of recombinant plasmids

1. Replace the small fragment between the SalI and PmlI sites of the pTSE-G1n vector (Beijing Baitemeibo Biological Co., Ltd.) with the DNA molecule shown in sequence 1 of the sequence listing to obtain a recombinant expression vector containing the variable region of the heavy chain (Verified by sequencing).

2. Replace the small fragment between the SalI and PmlI sites of the pTSE-K carrier (Beijing Baitemeibo Biological Co., Ltd.) with the DNA molecule shown in the sequence 3 of the sequence listing to obtain a recombinant expression vector containing the variable region of the light chain (Verified by sequencing).

2. Preparation of 3-3E Antibody

1. One day before transfection, FreeStyle ^TM HEK 293-F cells (Invitrogen, product number: R79007) were adjusted to a concentration of 1.0×10 ⁶ /ml, inoculated into a culture flask, and the cells were shaken at 37°C, 5% CO ₂ , and 125 rpm. Shake culture in bed for 24h.

2. After completing step 1, on the day of transfection, take the culture flask, add the transfection complex to the culture flask, shake and culture in a cell shaker at 37°C, 5% CO ₂ , and 125 rpm, and monitor cell viability after 48 hours. When the cell viability dropped to 80-85%, the culture supernatant was collected by centrifugation at 1,000 rpm for 10 min, and the antibody expression was detected by SDS-PAGE electrophoresis (lane 2 in Figure 1).

Transfection complex: Dilute 24 μl of FectoPRO transfection reagent in 3 ml of FreeStyle 293 medium (Gibco 12338-018), mix gently, and add 12 μg of the recombinant expression vector containing the variable region of the heavy chain prepared in step 1, containing light 12 μg of the recombinant expression vector of the chain variable region, mixed and placed at room temperature for 10 min.

3. After completing step 2, filter the supernatant with a 0.45 μm filter to remove impurities, add 10×PB to adjust the ion concentration to be close to the binding buffer; use AKTA purification system (GE, AKTA EXPLORER) for antibody purification, in AKTA Install the HiTrap MabSelect Xtra purification column in the purification instrument, set the corresponding system parameters, equilibrate the purification column with binding buffer and load the sample, then continue to equilibrate, and then rinse the prepacked column with citric acid solution (pH3.0) to elute the antibody protein , when the UV280 reaches 100, the collection starts, and when the UV280 drops to 100, the collection ends, and the buffer is replaced with a citrate solution (pH 6.0).

4. Take the antibody solution purified in step 3, and use SDS-PAGE electrophoresis to detect the expression of the antibody (lane 3 in Figure 1), and take samples to measure the protein concentration with a NanoDrop UV spectrophotometer (Thermo Scientific). The detected protein concentration is 1.25 mg/mL. .

Example 3, Binding ability detection of 3-3E antibody

1. Preparation of human adenovirus type 7 virus stock solution, virus concentrate and inactivated virus

1. Preparation of adenovirus virus solution

Adenovirus culture: conventional DMEM+10% (volume percentage) FBS medium to culture A549 cells (Peking Union Cell Resource Center, item number: 25), A549 cells were passaged in a 75cm ² cell flask the day before virus inoculation, so that The cell density reached 75% to 90% when the virus was inoculated on the second day; on the day of inoculation, slowly aspirate the cell culture medium in the culture flask, add 5ml DMEM to gently rinse the cells and discard, and add 3ml DMEM+2% (volume percent) content) FBS; use a micropipette to absorb an appropriate amount of HAdV7 virus into the cell bottle, infect at MOI ≈ 0.001, shake the bottle evenly several times to disperse the virus evenly, and place it in an incubator at 37°C, 5% CO ₂ for adsorption for 2h During the period, shake the culture flask every 30min or so; after the adsorption is completed, discard the virus culture medium, re-add 15ml of new DMEM+2% (volume percentage) FBS, and then place the cell flask at 37°C, 5% CO Continue to cultivate in the incubator of ₂ ; observe the cytopathic condition every day (cytopathic changes will appear after virus infection and proliferation, which is characterized by cell shrinkage, shedding, etc.); harvest when 75%-100% of cells have lesions, -80 ℃ Freeze and thaw twice, centrifuge at 4,000 rpm for 5 min, collect the supernatant, aliquot and store it at -80°C, which is the virus stock solution.

2. Preparation of adenovirus concentrate: The harvested virus culture was centrifuged at 4,000 rpm for 10 minutes to remove cell debris, and the supernatant was transferred to an ultrafiltration tube with a molecular weight cut-off of 50 kD (MILIPORE, product number UFC805008), and centrifuged at 4,000 rpm until the volume was reduced to 1/30 of the initial volume, collect the retentate, and store it at -80°C after aliquoting, which is the virus concentrate.

3. Adenovirus titer determination

One day before the experiment, A549 cells with good growth status (Peking Union Cell Resource Center, Cat. No. 25) were taken, and after trypsinization, the cell density was adjusted to 3×10 ⁵ /ml with DMEM+10% (volume percentage) FBS, and seeded on 96-well cell culture plate, 100 μl per well, cultured at 37°C, 5% CO ₂ ; take out the 96-well plate on the day of the experiment, discard the medium, wash it with serum-free medium, add DMEM+2% (volume percentage) FBS, 100 μl/well; then 10-fold gradient dilution of the virus solution to be tested in serum-free medium, 8 gradients from 10 ^-1 to 10 ^-8 in total, the diluted virus was added to the prepared 96-well plate at 10 μl/well, each Each dilution gradient was 8 wells, and a blank control group was set at the same time. After the operation, the cells were placed in an incubator at 37°C and 5% _CO2 for observation. After 7 days, the cell death in each well was counted, and the TCID50 of the virus stock solution was calculated according to the following formula.

Distance ratio = (percentage above 50% lesion rate - 50%)/(percentage above 50% lesion rate - percentage below 50% lesion rate)

LgTCID50 = difference between distance ratio × log dilution + log dilution above 50% lesion rate

After detection and calculation, the viral titer of the HAdV7 virus stock solution used in this study=5.0×10 ⁸ TCID50/ml, and the viral titer of the virus concentrate=1.0×10 ¹⁰ TCID50/ml.

4. Inactivation and purification of adenovirus

Take the virus stock solution and transfer it to a 500ml sample bottle, adjust the pH to 7.6 with sodium bicarbonate, then add β-propiolactone in a ratio of 1:2000, stir while adding, and after thorough mixing, continue stirring at 4°C for inactivation, 24 After 1 hour, adjust the pH to 7.6 again, add β-propiolactone according to the ratio of 1:2000, continue to stir and inactivate at 4 °C for 24 hours, take a sample of not less than 1‰ volume and hydrolyze it in a water bath at 37 °C for 4 hours (observed in the middle). When the color of the sample turns yellow, adjust the pH to about 7.0 with sodium bicarbonate. After the hydrolysis is completed, take the A549 cells transferred to the 25cm ² cell bottle the day before, and inoculate 1 bottle of 25cm ² A549 cells according to the ratio of 1ml of the sample (if less than 1ml, press 1ml At the same time, the cells inoculated with uninactivated virus and untreated empty cells were set as controls, placed in a 37°C, 5% _CO2 incubator for observation, and blindly transferred to new A549 cells after 7 days, and continued to observe, like this Blind passage for 3 generations, inoculated with uninactivated virus samples with cytopathic lesions and inactivated experimental group cells and blank cells with no lesions, it is judged that the inactivation test results are credible and the inactivation is complete; otherwise, the inactivation is incomplete and needs to be re-inactivated. Live, detect.

The virus stock solution confirmed to be completely inactivated by the inactivation test was centrifuged at 4,000 rpm for 10 minutes to remove cell debris, equilibrated on the Sepharose 4Fast Flow gel column with PBS buffer and loaded, and then eluted with PBS. The first elution peak is The target virus peak was collected, and then 12.5 ml of heavy density CsCl solution [42.23 g of cesium chloride + 57.77 ml of 10 mM Tris-HCl (pH 7.9- 8)], then slowly add 12.5ml of light density CsCl solution [22.39g of cesium chloride + 77.61ml of 10mM Tris-HCl (PH 7.9-8)], then add 15ml of virus suspension, balance it, and put it in ultracentrifugation In a machine (Beckman L100-XP), centrifugation was performed at 25,000 rpm and 4°C for 2 hours. The bands between light-density and heavy-density cesium chloride solutions were collected, dialyzed against PBS, and filter sterilized to obtain HAdV7 inactivated virus.

Samples were taken for routine SDS-PAGE detection and electron microscope observation. The preparation process of electron microscope samples is as follows: sampling 2.5% glutaraldehyde (0.075% PBS, pH7.4 dilution), fixed for 2 hours at 4 °C; Incubate for 5-10min; blot the liquid with filter paper; stain with 3% phosphotungstic acid (prepared with distilled water) for 2min at room temperature; blot dry with filter paper; observe on the machine.

The results of SDS-PAGE are shown in Figure 2, and the results of electron microscopy are shown in Figure 3.

2. 3-3E antibody specifically binds to human type 7 inactivated adenovirus

1. Take 4μl of the HAdV7 inactivated virus (200ng) prepared in step 1 of step 1, supplement with carbonate coating buffer (pH9.6) to 100ul, and add 100ul per well to the microtiter plate (Corning 9018), Three replicate wells were set up and coated overnight at 4°C.

2. After completing the above steps, take the ELISA plate, wash the plate three times with PBST, add PBS blocking solution containing 2% (mass percentage) BSA, and incubate at 37° C. for 2 hours.

3. After completing the above steps, take the ELISA plate, discard the blocking solution, add 100 μl of 3-3E antibody stock solution (concentration 300 μg/ml) to each well, incubate at 37°C for 90 min, and then PBST (PBS+0.1% Tween20) Wash the plate 3 times.

4. After completing the above steps, take the above enzyme-labeled plate, add 100 μl of 1:4000-fold diluted enzyme-labeled antibody (goat anti-human IgG-HRP, Zhongshan Jinqiao, product number ZB-2304) to each well, incubate at 37°C for 45 minutes, and then PBST (PBS+0.1% Tween20) washes the plate 3 times.

5. After completing the above steps, take the ELISA plate, add 50 μl of OPD substrate chromogenic solution to each well, and incubate at room temperature for 10 minutes.

6. After completing the above steps, take the ELISA plate and add 50 μl of 1M sulfuric acid solution to each well to terminate the enzyme-linked reaction.

7. Microplate reader 492nm/630nm dual wavelength to measure optical density value.

In the above steps, an inactivated influenza virus antigen (influenza virus A/swine/Colorado/1/77 (H3N2) was replaced by the HAdV7 virus, prepared according to the method of step 1) was set as a control group for the antigen to replace the HAdV7 inactivated virus.

The results are shown in Figure 4. The results showed that the 3-3E antibody could specifically bind to inactivated adenovirus but not to influenza virus (Flu).

3. Analysis of antigen-binding ability of 3-3E antibody

1. Take 4 μl of the HAdV7 inactivated virus (200ng) prepared in step 1, 4, supplement with carbonate coating buffer (pH 9.6) to 100 μl, and add 100 μl per well to the microtiter plate (Corning product number: 9018) , set up 3 duplicate wells, and coat overnight at 4°C.

2. After completing the above steps, take the ELISA plate, wash the plate three times with PBST, add PBS blocking solution containing 2% (mass percentage) BSA, and incubate at 37° C. for 2 hours.

3. After completing the above steps, take the ELISA plate, discard the blocking solution, and add 100 μl of 3-3E antibody solution (initial concentration 400 μg/ml) diluted by 2 times equal ratio to each well, setting a total of 30 gradients, After incubation at 37°C for 90 min, the plate was washed three times with PBST.

4. After completing the above steps, take the ELISA plate, add 100 μl of 1:4000-fold diluted HRP-labeled anti-human IgG antibody (Zhongshan Jinqiao, product number ZB-2304) to each well, incubate at 37°C for 45 minutes, and wash the plate with PBST for 3 Second-rate.

5. After completing the above steps, take the ELISA plate, add 50 μl of OPD substrate chromogenic solution to each well, and incubate at room temperature for 10 minutes.

6. After completing the above steps, take the ELISA plate and add 50 μl of 1M sulfuric acid solution to each well to terminate the enzyme-linked reaction.

7. Microplate reader 492nm/630nm dual wavelength to measure optical density value.

The results are shown in Figure 5. In Figure 5, the abscissa is the logarithmic value of the protein concentration, and the ordinate is the optical density value. Analysis showed that the binding ability of 3-3E antibody to human adenovirus type 7 was EC50=339.3 nM.

Example 4. The efficacy of 3-3E against human adenovirus type 7 infection

1. In vitro detection of the preventive and therapeutic effects of 3-3E against human adenovirus type 7 infection

1. One day before the experiment, take well-grown A549 cells, digest them with trypsin, adjust the cell density to 3×10 ⁵ /ml with DMEM+10% (volume percentage) FBS, and inoculate them in a 96-well cell culture plate. 100 μl, incubate at 37°C with 5% CO ₂ .

2. After completing step 1, take out the 96-well plate on the day of the experiment, discard the medium, wash it with serum-free medium, and add DMEM+2% (volume percent) FBS, 100 μl per well; then group the following operations:

Experimental group (3-3E 0h): The 3-3E antibody prepared in Example 2 was diluted with serum-free medium to obtain different concentrations (50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.2 μg/ml, 1.6μg/ml, 0.8μg/ml, 0.4μg/ml, 0.2μg/ml) 3-3E antibody test antibody solution; the test antibody solution and the test antibody solution prepared in step 1 of Example 3 The HAdV7 virus stock solution (diluted with serum-free medium to a virus concentration of 2×10 ³ TCID50/mL) was mixed according to the volume ratio of 1:1, incubated at 37°C for 1.5h, added to the well, and kept at 37°C, 5% CO2 Incubate for 1 h in the incubator.

Experimental group (3-3E+1h): The 3-3E antibody prepared in Example 2 was diluted with serum-free medium to obtain different concentrations ((50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.2μg/ml, 1.6μg/ml, 0.8μg/ml, 0.4μg/ml, 0.2μg/ml) 3-3E antibody test antibody solution; The HAdV7 virus stock solution ( Dilute it with serum-free medium to a virus concentration of 2×10 ³ TCID50/mL) and add it to the well. After incubating at 37°C for 1 h, add the same volume of the antibody solution to be tested as the virus solution to the well, and continue to place it at 37°C. , and incubated for 1 h in a 5% CO2 incubator.

Irrelevant antibody control group (Anti-DENV1): Anti-DENV1 antibody (documented in the literature: PotentNeutralization Ability of a Human Monoclonal Antibody Against Serotype1Dengue Virus. Frontiers in Microbiology, 1June2018, Volume 9, Article 1214; the public can obtain from the Chinese People's Liberation Army Military obtained from the Academy of Military Medical Sciences of the Academy of Sciences) diluted with serum-free medium to obtain different concentrations (25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.2 μg/ml, 1.6 μg/ml, 0.8 μg/ml, 0.4 μg /ml, 0.2μg/ml and 0.1μg/ml) Anti-DG antibody test antibody solution; the test antibody solution and the HAdV7 virus stock solution prepared in step 1 of Example 3 (1 diluted with serum-free medium to The virus concentration was 2×10 ³ TCID50/mL) and mixed according to the volume ratio of 1:1, incubated at 37°C for 1.5h, added to the well, and continued to incubate for 1h in a 37°C, 5% CO2 incubator.

Irrelevant antibody control group (Anti-EGFR): Anti-EGFR antibody (recorded in invention patent CN102993305B) was diluted with serum-free medium to obtain different concentrations (25μg/ml, 12.5μg/ml, 6.25μg/ml, 3.2 μg/ml, 1.6μg/ml, 0.8μg/ml, 0.4μg/ml, 0.2μg/ml and 0.1μg/ml) Anti-DG antibody test antibody solution; The HAdV7 virus stock solution prepared in 1 (diluted with serum-free medium to a virus concentration of 2×10 ³ TCID50/mL) was mixed according to the volume ratio of 1:1, incubated at 37°C for 1.5h, added to the well, and placed in 37 Incubate for 1 h in a 5% CO2 incubator.

Virus group (VIRUS): Serum-free medium and the HAdV7 virus stock solution prepared in step 1 of Example 3 (diluted with serum-free medium to a virus concentration of 2×10 ³ TCID50/mL) in a volume ratio of 1:1 Mix, incubate at 37°C for 1.5h, add to the well, 100 μl/well, and continue to incubate for 1h in a 37°C, 5% CO ₂ incubator.

Empty cell control group (CELL): serum-free medium, incubated at 37°C for 1.5h, added to the well, and then incubated at 37°C in a 5% CO2 incubator for 1 hour.

3. After completing step 2, take a 96-well plate, discard the supernatant, add DMEM+2% (volume percentage) FBS medium (100 μl/well), and continue to culture at 37°C, 5% CO2 for 1 week until cells appear. Obvious lesions; take out the culture plate, observe under a microscope, count the number of cells with no lesions, partial lesions and complete lesions, and calculate the inhibition rate of antibodies against adenovirus infection (Table 1).

Table 1 Detection results of 3-3E anti-HAdV7 infected cells

*Concentration unit: μg/mL; 0h: inoculate cells after co-incubating antibody and virus; +1h: add antibody 1 hour after virus infection

The results show that the antibody 3-3E prepared by the present invention can effectively inhibit the proliferation of HAdV7, with EC50=0.4 μg/mL for preventive use, and EC50=0.72 μg/mL for use after virus infection.

2. 3-3E anti-human adenovirus type 7 infects CB-17SCID mice

1. Establishment of animal models

Take CB-17SCID mice (Vitron Lever), female, 16-18g, weighed on the day of the experiment, and randomly divided into experimental group and control group (12 mice in each group), isoflurane inhalation anesthesia, and the experimental group was inoculated by nasal drip The HAdV7 virus concentrate (10 ⁸ TCID50/piece) prepared in step 1, step 2 of Example 3, the control group was inoculated with an equal volume of culture medium intranasally. The body weight was measured regularly every day and the hair loss, eating, drinking and activity of the test animals were observed.

The results are shown in Figure 6. The results showed that the mice in the experimental group started to eat less and drink less water the next day after inoculation with the virus, their hair became rough, and the body was thin. .

2. Effect of Antibody 3-3E Antibody on Uninfected Mice

Take CB-17SCID mice (Vitron Lever), female, 16-18g, weighed on the day of the experiment, and randomly divided into experimental group and control group (12 mice in each group), isoflurane inhalation anesthesia, and Example 2 was prepared The 3-3E antibody was diluted with serum-free DMEM medium to obtain an antibody solution containing 200 mg/ml 3-3E antibody. The experimental group was instilled with 20 μl nasally, and the control group was inoculated with an equal volume of serum-free medium. The body weight was measured regularly every day and the hair loss, eating, drinking and activity of the test animals were observed.

The results are shown in Figure 7. The results showed that there was no significant change in the body of the experimental group compared with the control group, and the body weight continued to increase, confirming that the antibody inoculation of the experimental group would not have a significant effect on the mice.

2. 3-3E anti-human adenovirus type 7 infection of CB-17SCID mice

Take CB-17SCID mice (Vitron Lever), female, 16-18g, weighed on the day of the experiment, and randomly divided into experimental group and control group (12 mice in each group) for the following operations:

Experimental group (3-3E): The prepared 3-3E antibody was diluted with serum-free DMEM medium to obtain an antibody solution containing 200ug/ml 3-3E antibody; the antibody solution was mixed with the HAdV7 prepared in step 2 of Example 3 The virus concentrate (the virus concentration is 10 ¹⁰ TCID50/ml) was mixed according to the volume ratio of 1:1, and incubated at 37°C for 1 h. The mice in the experimental group prepared in step 1 were intranasally inoculated with 20 μl each, and the mice were observed for hair loss, eating, Drink water and activities and record the body weight of mice.

Control group (VIRUS-only): The HAdV7 virus concentrate prepared in step 2 of Example 3 (the virus concentration is 10 ¹⁰ TCID50/ml) was mixed with serum-free DMEM medium at a volume ratio of 1:1, and incubated at 37°C For 1 h, the mice in the experimental group prepared in step 1 were inoculated intranasally (the inoculation volume of each mouse was 20 μl), and the hair loss, eating, drinking and activity of the mice were observed, and the weight of the mice was recorded.

The results are shown in Figure 8. The results showed that the control group showed new and obvious symptoms such as weight loss and rough coat color on the second day, while the symptoms of the mice in the experimental group were significantly reduced compared with the control group, indicating that the 3-3E antibody can effectively reduce the weight loss caused by the virus infection in the mice. and rough coat symptoms.

sequence listing

<110> Academy of Military Medicine, Academy of Military Sciences, Chinese People's Liberation Army

<120> Human adenovirus type 7 monoclonal antibody 3-3E and its application

<160> 4

<170> SIPOSequenceListing 1.0

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<211> 354

<212> DNA

<213> Artificial Sequence

<400> 1

caggtgcagc tggtggagtc tgggggaggc tcggtccagc cgggggggtc cctgagactc 60

tcctgtgcag tctctggatt cacctttagt agttattgga tgagctgggt ccgccaggct 120

ccagggaaga ggctggagtg ggtggccaac ataaacgaag atggaagtga gaaagacttt 180

gtggactctg tgaagggccg attcaccatc tccagaggca acaccaagaa ctcactgtat 240

ctgcaaatga gcagcctgag aaccgaggac acggctgtgt actactgtgc gaggggccag 300

agagggcagt ggctggccct ctttgactac tggggccaag ggaccacggt cacc 354

<210> 2

<211> 121

<212> PRT

<213> Artificial Sequence

<400> 2

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val

35 40 45

Ala Asn Ile Asn Glu Asp Gly Ser Glu Lys Asp Phe Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Gly Asn Thr Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gln Arg Gly Gln Trp Leu Ala Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 3

<211> 324

<212> DNA

<213> Artificial Sequence

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gacatccaga tgacccagtc tccggcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc ggacaagtca gagcattagc accttcttaa attggtatca gcacaaagca 120

ggaagcgccc ctaatctctt gatctatgct gcatccagtt tgcagagtgg agtcccatca 180

agattcagtg gcagtggatc tgggacagtt ttcactctca ccattagcgg tctgcaacct 240

gaagattctg caacttacta ctgtcaacag agttacagca acccctcttt cggcggaggg 300

accaaggtgg agatcaaacg aact 324

<210> 4

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Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Thr Phe

20 25 30

Leu Asn Trp Tyr Gln His Lys Ala Gly Ser Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Val Phe Thr Leu Thr Ile Ser Gly Leu Gln Pro

65 70 75 80

Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Asn Pro Ser

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr

100 105

Claims (8)

1. a monoclonal antibody against human type 7 adenovirus, comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions HCDR1, HCDR2 and HCDR3; The chain variable region includes three complementarity determining regions LCDR1, LCDR2 and LCDR3; The amino acid sequence of the HCDR1 is shown in the 26th-33rd position from the N-terminus of Sequence 2 of the Sequence Listing; The amino acid sequence of the HCDR2 is shown in the 51-58th position from the N-terminus of Sequence 2 of the Sequence Listing; The amino acid sequence of the HCDR3 is shown in the 97th-110th position from the N-terminus of Sequence 2 of the Sequence Listing; The amino acid sequence of the LCDR1 is shown in the 27th-32nd position from the N-terminus of Sequence 4 of the Sequence Listing; The amino acid sequence of the LCDR2 is shown in the 50th-52nd position from the N-terminus of Sequence 4 of the Sequence Listing; The amino acid sequence of LCDR3 is shown in the 89th-96th position from the N-terminus of Sequence 4 of the Sequence Listing. 2. The monoclonal antibody against human adenovirus type 7 as claimed in claim 1, wherein: The amino acid sequence of the heavy chain variable region is shown in Sequence 2 of the sequence listing; The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4 of the sequence listing. 3. A gene encoding the monoclonal antibody against human adenovirus type 7 according to claim 1 or 2. 4. The gene of claim 3, wherein: The gene encoding the variable region of the heavy chain of the antibody is the DNA molecule shown in Sequence 1 of the sequence listing; The gene encoding the light chain variable region of the antibody is the DNA molecule shown in SEQ ID NO: 3 of the sequence listing. 5. Use of the anti-human adenovirus type 7 monoclonal antibody of claim 1 or 2 in the preparation of a medicament for preventing and/or treating diseases caused by human adenovirus type 7 infection. 6. The application of the anti-human adenovirus type 7 monoclonal antibody of claim 1 or 2 in the preparation of a product; the use of the product is as follows (e1) and/or (e2): (e1) inhibition of human adenovirus type 7; (e2) Neutralization of human adenovirus type 7. 7 . A medicine for preventing and/or treating diseases caused by human adenovirus type 7 infection, the active ingredient is the monoclonal antibody against human adenovirus type 7 according to claim 1 or 2 . 8. A product whose active ingredient is the monoclonal antibody against human adenovirus type 7 according to claim 1 or 2; the use of the product is as follows (e1) and/or (e2): (e1) inhibition of human adenovirus type 7; (e2) Neutralization of human adenovirus type 7.

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