CN109897104B

CN109897104B - Human adenovirus 7 monoclonal antibody 3-3E and application thereof

Info

Publication number: CN109897104B
Application number: CN201910242181.1A
Authority: CN
Inventors: 杨志新; 王荣; 陆健昇; 余云舟; 周权; 周晓巍
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: Institute of Pharmacology and Toxicology of AMMS
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2020-12-22
Anticipated expiration: 2039-03-28
Also published as: CN109897104A; WO2020192080A1

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 7 monoclonal antibody 3-3E and application thereof

Technical Field

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

Background

Human adenovirus belongs to the genus adenovirus of mammals belonging to the family adenoviridae, and was first discovered in 1953, and was isolated and cultured from amygdala-like tissues atrophic from healthy people; the genome of the virus is double-stranded DNA with the total length of about 36kb, the double-stranded DNA is combined with virus structural proteins to form a virus core, the virus is not enveloped, the core is coated with a capsid, the capsid is composed of 252 capsids, 240 of the capsids are hexon proteins and 12 penton proteins, the capsid is in a regular 20-face structure, and the diameter is about 80-110 nm.

The adenoviruses that have been found to date have 7 subgroups, 67 distinct serotypes, of which 55 subtypes are capable of infecting and causing disease in humans. Adenovirus infection is most common to people infected with respiratory tract and causes respiratory diseases, and besides, partial viruses can also cause urinary system and gastrointestinal system infection, and researches show that adenovirus causing respiratory tract infection mainly comprises A, C, E and B1 groups, wherein the B1 subgroup, the B2 subgroup can infect the urinary system of people, and the F, D group can infect the gastrointestinal system and colonic system of people respectively. Currently, adenovirus causing respiratory tract infection in the world is known to be 1, 2, 3, 4, 7, 14 and 55 types of B1 subgroups, wherein 3 types, 4 types, 7 types and 14 types are the most common types causing outbreak epidemic, and once caused outbreak epidemic of respiratory tract adenovirus in Jiangsu and Taiwan provinces in China, Korea, Singapore, Malaysia and the like. Since 2008, many outbreak infectious diseases spread through respiratory tracts occur successively in different areas of China, the epidemic situation spread is wide, the infectivity is strong, and the outbreak infectious diseases are respectively B group 7 type adenovirus, 55 type adenovirus and 14 type adenovirus through laboratory pathogen detection and identification.

Generally, when a human body is infected by viruses, the immune system of the human body can stimulate humoral immunity and cellular immune response and gradually control infection and remove the viruses, IgM in the body of a patient begins to be generated about 3 days after the infection of most viruses to the human body causes diseases, most of IgG begins to be generated at 7-10 days later, then the IgG gradually rises, and the peak is reached about 1 month generally. Therefore, the neutralizing antibody plays an important role in the recovery of virus infection all the time, and the adenovirus infected human body can also induce stronger humoral immune response to generate specific antibodies. Research shows that the organism can generate effective immunity to the reinfection of homotype adenovirus and can generate long-time immune protection (more than 10 years), the reinfection can not be generated after recovery generally, and the neutralizing antibody generated by the organism is generated in the process of the adenovirus infection, according to the research, 40-60% of people in 6-15 years old in China have the neutralizing antibodies of

types

1, 2 and 5, and the antibody of a mother can protect the baby from serious adenovirus infection. Therefore, the adenovirus neutralizing antibody is considered to be an effective and specific anti-adenovirus therapeutic means, and the development of the adenovirus neutralizing antibody should be a main development direction for the development of future adenovirus infection therapeutic drugs.

Currently, no specific therapeutic drug aiming at adenovirus is used in clinic in the world, drug development is mostly limited to chemical drugs, namely nucleoside analogues resisting DNA virus, and research on specific biological drugs aiming at adenovirus is blank.

Disclosure of Invention

The invention aims to provide a human adenovirus 7 monoclonal antibody 3-3E and application thereof.

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

(a1) an amino acid sequence shown in 26 th-33 th site from N end of the sequence 2in the sequence table;

(a2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of an amino acid sequence shown in 26 th to 33 th sites of the N end of the sequence 2in the sequence table;

(a3) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown in the 26 th to 33 th sites from the N end of the sequence 2 of the sequence table and has the same function;

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

(a4) an amino acid sequence shown in the 51 st-58 th site from the N end of the sequence 2in the sequence table;

(a5) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of an amino acid sequence shown in the 51 th to 58 th sites of the N end of the sequence 2in the sequence table;

(a6) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown by the 51 th to 58 th sites from the N end of the sequence 2in the sequence table and has the same function;

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

(a7) an amino acid sequence shown in 97 th-110 th site from the N end of the sequence 2in the sequence table;

(a8) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues to an amino acid sequence shown from 97 th to 110 th sites of the N end in the sequence 2 of the sequence table;

(a9) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown by 97 th-110 th site from the N end of the sequence 2 of the sequence table and has the same function;

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

(b1) an amino acid sequence shown in 27 th to 32 th positions from the N end of a sequence 4 in the sequence table;

(b2) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of an amino acid sequence shown in 27 th to 32 th sites from the N end of the sequence 4 in the sequence table;

(b3) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown in the 27 th to 32 th sites from the N end of the sequence 4 of the sequence table and has the same function;

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

(b4) an amino acid sequence shown in the 50 th-52 th site from the N end of the sequence 4 in the sequence table;

(b5) amino acid sequences with the same function are obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequences shown in the 50 th to 52 th sites from the N end of the sequence 4 in the sequence table;

(b6) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown by the 50 th to 52 th sites from the N end of the sequence 4 of the sequence table and has the same function;

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

(b7) an amino acid sequence shown in 89 th to 96 th sites from the N end of the sequence 4 in the sequence table;

(b8) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues of an amino acid sequence shown in 89 th to 96 th sites from the N end of the sequence 4 in the sequence table;

(b9) and (b) an amino acid sequence which has homology of 75% or more than 75% with the amino acid sequence shown in the 89 th-96 th site from the N end of the sequence 4 of the sequence table and has the same function.

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

(c1) an amino acid sequence shown in a sequence 2in a sequence table;

(c2) the amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 of the sequence table;

(c3) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown in the sequence 2 of the sequence table and has the same function;

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

(c4) an amino acid sequence shown as a sequence 4 in a sequence table;

(c5) an amino acid sequence with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 4 of the sequence table;

(c6) an amino acid sequence which has 75 percent or more than 75 percent of homology with the amino acid sequence shown in the sequence 4 of the sequence table and has the same function.

The invention also protects the gene encoding the antibody.

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

(d1) a DNA molecule shown in a sequence 1 of a sequence table;

(d2) a DNA molecule having 75% or more homology with the nucleotide sequence defined in (d1) and encoding the heavy chain variable region as set forth in

claim

1 or 2;

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

claim

1 or 2;

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

(d4) a DNA molecule shown in a sequence 3 of a sequence table;

(d5) a DNA molecule having 75% or more homology with the nucleotide sequence defined in (d4) and encoding the heavy chain variable region as set forth in

claim

1 or 2;

(d6) a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in (d4) or (d5) and encodes the heavy chain variable region of

claim

1 or 2.

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

The invention also protects the application of any monoclonal antibody in preparing products; the application of the product is (e1) and/or (e 2):

(e1) inhibiting adenovirus;

(e2) and (3) neutralizing the adenovirus.

The invention also provides a medicament for preventing and/or treating diseases caused by adenovirus infection, and the active ingredient of the medicament is the monoclonal antibody.

The invention also protects a product, the active ingredient of which is the monoclonal antibody described in any one of the above; the application of the product is (e1) and/or (e 2):

(e1) inhibiting adenovirus;

(e2) and (3) neutralizing the adenovirus.

Any of the above adenoviruses is a human adenovirus. The human adenovirus is human adenovirus 7.

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.

Drawings

FIG. 1 shows the expression and purification assays for 3-3E.

FIG. 2 shows SDS-PAGE electrophoretic detection of HAdV 7.

FIG. 3 is an electron microscope examination of HAdV 7.

FIG. 4 is an antigen specificity assay for 3-3E.

FIG. 5 is an affinity assay of 3-3E.

FIG. 6 shows the construction of animal models infected with adenovirus.

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

FIG. 8 is a graph of the protective effect of 3-3E on HAdV7 infected CB-17SCID mice.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

HAdV7 virus: human/CHN/GZ6965/2001, described in the literature: yu Z, Zeng Z, Zhang J, et al.facial Community-acquired Pneumonia in Children used by Re-emergent Human Adenovir 7d Associated with high ther specificity of Illness and Fatality Rate [ J ]. Scientific Reports,2016,6: 37216.; the public can be obtained from the military medical research institute of military science institute of the national people liberation military.

Influenza virus antigens: A/brine/Colorado/1/77 (H3N2), described in the literature: karasin AI, Schuten M, Cooper LA, et al, genetic characterization of H3N2 underfluence viruses isolated from seeds in North America,1977-1999, evaluation for porous human and inorganic viruses Research [ J ]. Virus Research,2000,68(1): 71-85.; the public can be obtained from the military medical research institute of military science institute of the national people liberation military.

Example 1 discovery of antibodies

Collecting peripheral blood of a DENV-1 infected person in a convalescent period, and separating and collecting peripheral blood mononuclear lymphocytes from the peripheral blood. The collected peripheral blood mononuclear lymphocytes are labeled by adding corresponding molecular marker antibodies (BD, #555332, #555415, #555441, #560677, #555622) or control antibodies (BD, #555748, #555742, #555751, #555749, #557872) on the surfaces of the cells, and then flow sorting is carried out, and the sorted cells are used for antibody gene amplification. Using the separated single B cell as a template, amplifying antibody genes, sequencing and expressing the positively amplified clones, using a 7-type adenovirus purified sample as an antigen, and obtaining 1 antibody sequence through mass screening, analysis and verification, wherein the sequence is named as a 3-3E antibody.

The amino acid sequence of the heavy chain variable region of the 3-3E antibody is shown as a sequence 2in a sequence table (wherein, the 26 th-33 th amino acid residues from the N end form a CDR1, the 51 th-58 th amino acid residues form a CDR2, and the 97 th-110 th amino acid residues form a CDR3), and the coding gene thereof is shown as a sequence 1 in the sequence table.

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

EXAMPLE 2, 3-3E antibody preparation

Construction of recombinant plasmid

1. A small fragment between SalI and PmlI sites of a pTSE-G1n vector (Beijing Baite Meibo biology, Ltd.) was replaced by a DNA molecule shown in sequence 1 of the sequence listing to obtain a recombinant expression vector containing a heavy chain variable region (verified by sequencing).

2. A small fragment between SalI and PmlI sites of a pTSE-K vector (Beijing Baitemeibo biology, Ltd.) is replaced by a DNA molecule shown in a sequence 3 of a sequence table to obtain a recombinant expression vector containing a light chain variable region (sequencing verification is carried out).

Preparation of Di, 3-3E antibodies

1. FreeStyle one day before transfection^TMHEK 293-F cells (Invitrogen, cat # R79007) were adjusted to a concentration of 1.0X 10⁶/ml, inoculated into a culture flask, at 37 ℃ with 5% CO₂Shaking and culturing the cells in a 125rpm cell shaking table for 24 hours.

2. On the day of transfection after completion of step 1, the flask was taken and the transfection complex was added to the flask at 37 ℃ with 5% CO₂Shaking the cells in a shaker at 125rpm, monitoring the cell activity at 48 hours, centrifuging the cells at 1,000rpm for 10min when the cell activity decreases to 80-85%, and determining the antibody expression by SDS-PAGE (lane 2in FIG. 1).

Transfection complex: mu.l of FectoPRO transfection reagent was diluted in 3ml of FreeStyle 293 medium (Gibco 12338-018), gently mixed, and 12. mu.g of the recombinant expression vector containing the heavy chain variable region prepared in step one and 12. mu.g of the recombinant expression vector containing the light chain variable region were added, and after mixing, they were left at room temperature for 10 min.

3. After step 2 is completed, filtering the supernatant by using a 0.45-micron filter membrane to remove impurities, and adding 10 XPB to adjust the ion concentration to be similar to that of the binding buffer solution; antibody purification was performed using the AKTA purification system (GE, AKTA EXPLORER), a HiTrap MabSelect Xtra purification column was installed in the AKTA purification apparatus, the corresponding system parameters were set, the purification column was equilibrated with binding buffer and loaded, then equilibration was continued, then the pre-packed column was washed with citric acid solution (ph3.0) to elute antibody proteins, collection started when UV280 reached 100, collection ended when UV280 decreased to 100, and the buffer was replaced with citrate solution (ph 6.0).

4. The antibody solution purified in step 3 was taken, subjected to SDS-PAGE to detect antibody expression (lane 3 of FIG. 1), and sampled to determine the protein concentration by a NanoDrop ultraviolet spectrophotometer (Thermo Scientific), wherein the protein concentration was 1.25 mg/mL.

Example 3, 3-3E antibody binding Capacity test

Preparation of human 7 type adenovirus virus stock solution, virus concentrated solution and inactivated virus

1. Preparation of adenovirus virus liquid

Culturing adenovirus: a549 cells (Beijing coordination cell resource center, cat # 25) were cultured in conventional DMEM + 10% (volume percent) FBS medium, and A549 cells were passaged to 75cm on the day before virus inoculation²In the cell bottle, the cell density reaches 75-90% when the virus is inoculated on the next day; on the day of inoculation, slowly sucking out the cell culture medium in the culture bottle, adding 5ml of DMEM, slightly washing the cells, discarding the cells, and adding 3ml of DMEM + 2% (volume percentage content) FBS; sucking appropriate amount of HAdV7 virus with micropipette into cell bottle, infecting with MOI of about 0.001, shaking the bottle for several times to disperse the virus, standing at 37 deg.C and 5% CO₂Adsorbing for 2h in the incubator, and shaking the culture bottle once every 30 min; after adsorption was complete, the virus culture medium was discarded, 15ml of fresh DMEM + 2% (volume percent) FBS was added again, and the cell vial was placed at 37 ℃ with 5% CO₂Incubator relayContinuing culturing; observing cytopathic conditions every day (cytopathic conditions appear after virus infection and proliferation, and are characterized by cell shrinkage, cell shedding and the like); harvesting when 75-100% of cells have pathological changes, freezing and thawing at-80 ℃ for 2 times, centrifuging at 4,000rpm for 5min, collecting supernatant, subpackaging, and storing at-80 ℃ to obtain virus stock solution.

2. Preparing an adenovirus concentrated solution: the harvested virus culture was centrifuged at 4,000rpm for 10 minutes to remove cell debris, the supernatant was transferred to an ultrafiltration tube (MILIPORE, cat # UFC805008) with a cut-off molecular weight of 50kD, centrifuged at 4,000rpm to 1/30 where the volume was reduced to the initial volume, the cut-off was collected, and the virus concentrate was stored at-80 ℃ after split charging.

3. Adenovirus titer determination

A549 cells (Beijing coordination cell resource center, cat # 25) with good growth state are taken one day before the experiment, and the cell density is adjusted to 3 x 10 by DMEM + 10% (volume percentage content) FBS after the pancreatin digestion⁵Perml, inoculated in 96-well cell culture plates at 100. mu.l/well, 37 ℃ with 5% CO₂Culturing; taking out a 96-well plate on the same day of the experiment, abandoning the culture medium, washing the serum-free culture medium once, and adding DMEM + 2% (volume percentage content) FBS (fetal bovine serum) into the culture medium at a concentration of 100 mu l/well; then diluting the virus solution to be tested with serum-free medium by 10-fold gradient, 10^-1～10^-8Adding diluted virus into prepared 96-well plate according to 10 μ l/well with 8 wells per dilution gradient, setting blank control group, placing the cells at 37 deg.C and 5% CO₂The cell death of each well was counted after 7 days, and TCID50 of the virus stock was calculated according to the following equation.

Distance scale ═ (percentage above 50% variability-50%)/(percentage above 50% variability-percentage below 50% variability)

lgTCID50 ═ distance proportion x difference between log of dilutions + log of dilutions above 50% disease rate

The virus titer of the HAdV7 virus stock used in this study was determined and calculated to be 5.0 × 10⁸TCID50/ml, virus titer of virus concentrate 1.0 × 10¹⁰TCID50/ml。

4. Inactivation and purification of adenoviruses

Transferring virus stock solution to a 500ml sample bottle, adjusting pH to 7.6 with sodium bicarbonate, adding beta-propiolactone according to the proportion of 1:2000 while stirring, mixing uniformly, continuously stirring and inactivating at 4 ℃, adjusting pH to 7.6 again after 24 hours, supplementing beta-propiolactone according to the proportion of 1:2000, continuously stirring and inactivating at 4 ℃ for 24 hours, hydrolyzing the sample with the volume of not less than 1 per thousand in a water bath at 37 ℃ for 4 hours (adjusting pH to about 7.0 with sodium bicarbonate when the color of the sample is observed to turn yellow), finishing hydrolysis, and transferring to 25cm in the previous day²A549 cells from cell flask, 1 flask and 25cm were inoculated with 1ml of sample²A549 cells were inoculated at a ratio (less than 1ml based on 1 ml) while the cells inoculated with non-inactivated virus and untreated empty cells were set as controls and placed at 37 ℃ in 5% CO₂And (3) carrying out culture observation in the incubator, carrying out blind transfer to new A549 cells after 7 days, carrying out continuous observation, carrying out blind transfer for 3 generations, inoculating cells of the inactivated virus sample with pathological changes, judging that the inactivated detection result is credible and the inactivation is complete, and otherwise, carrying out incomplete inactivation and needing to be inactivated and detected again.

The virus stock solution which is completely inactivated is verified by inactivation detection, cell debris is removed by centrifugation at 4,000rpm for 10 minutes, a Sepharose 4Fast Flow gel column is equilibrated by PBS buffer solution and loaded, then PBS is used for elution, the first elution peak is the target virus peak, the elution peak is collected, then 12.5ml of CsCl solution with heavy density [ 42.23g of cesium chloride +57.77ml of 10mM Tris-HCL (pH 7.9-8) ] is added into an Amicon-Ultra-15 ultrafiltration tube (50kD), 12.5ml of CsCl solution with light density [ 22.39g of cesium chloride +77.61ml of 10mM Tris-HCL (pH 7.9-8) ] is slowly added, 15ml of virus suspension is added, the mixture is leveled, and the mixture is placed in an ultracentrifuge (Beckman L100-XP) and centrifuged at 25,000rpm and 4 ℃ for 2 hours. Bands between light and heavy density cesium chloride solutions were collected, dialyzed against PBS, and filter sterilized to give HAdV7 inactivated virus.

Samples were taken for routine SDS-PAGE detection and electron microscopy, respectively. The preparation process of the electron microscope sample comprises the following steps: 2.5% glutaraldehyde (0.075% PBS, diluted at pH 7.4) was sampled and fixed at 4 ℃ for 2 hours; then taking 15-20ul of liquid to be detected, dripping the liquid to a copper net (containing a carbon supporting membrane), and incubating for 5-10min at room temperature; sucking the liquid through filter paper; dyeing 3% phosphotungstic acid (prepared by distilled water) at room temperature for 2 min; sucking the liquid through filter paper; and (5) operating the machine for observation.

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

II, 3-3E antibody specific binding human 7 type inactivated adenovirus

1. Mu.l of the HAdV7 inactivated virus (200ng) prepared in step one, 4, supplemented with carbonate coating buffer (pH 9.6) to 100ul, was added to an enzyme plate (Corning 9018) at 100ul per well, 3 wells were set, and coated overnight at 4 ℃.

2. After the above steps are completed, the ELISA plate and the PBST plate are washed for 3 times, PBS blocking solution containing 2% (mass percentage content) BSA is added, and incubation is carried out for 2 hours at 37 ℃.

3. After the above steps are completed, the microplate is removed, the blocking solution is discarded, 100. mu.l of 3-3E antibody stock (concentration 300. mu.g/ml) is added to each well, incubation is carried out at 37 ℃ for 90min, and then the plate is washed 3 times with PBST (PBS + 0.1% Tween 20).

4. After the above steps are completed, the enzyme label plate is taken, and 100. mu.l of 1: the enzyme-labeled antibody (goat anti-human IgG-HRP, China fir gold bridge, cat # ZB-2304) was diluted 4000 times, incubated at 37 ℃ for 45min, and then washed 3 times with PBST (PBS + 0.1% Tween 20).

5. After the above steps are completed, the ELISA plate is taken, 50 μ l of OPD substrate developing solution is added into each hole, and incubation is carried out for 10 minutes at room temperature.

6. After the above steps are completed, the ELISA plate is taken, and 50 μ l of 1M sulfuric acid solution is added into each hole to terminate the enzyme-linked reaction.

7. The optical density value is measured by a microplate reader 492nm/630nm double wavelength.

The steps are simultaneously provided with a control group which adopts an inactivated influenza virus antigen (prepared by replacing the influenza virus A/swine/Colorado/1/77(H3N2) with HAdV7 virus according to the method of the step one) as an antigen to replace HAdV7 inactivated virus.

The results are shown in FIG. 4. The results indicate that the 3-3E antibody can specifically bind to inactivated adenovirus and not to influenza virus (Flu).

Analysis of antigen binding Capacity of Tri, 3-3E antibody

1. Mu.l of the HAdV7 inactivated virus (200ng) prepared in step one, 4, supplemented with carbonate coating buffer (pH 9.6) to 100. mu.l, was added to an enzyme plate (Corning Commodity No.: 9018) at 100. mu.l per well, and 3 wells were set and coated overnight at 4 ℃.

3. After the above steps are completed, the enzyme label plate is taken, the blocking solution is discarded, 100 microliter of 3-3E antibody liquid (the initial concentration is 400 microgram/ml) diluted according to 2 times of equal ratio is added into each hole, 30 gradients are set in total, incubation is carried out for 90min at 37 ℃, and then the plate is washed for 3 times by PBST.

4. After the above steps are completed, the microplate is taken, and 100. mu.l of 1: the HRP-labeled anti-human IgG antibody (China fir gold bridge, cat # ZB-2304) was diluted 4000 times, incubated at 37 ℃ for 45min, and washed 3 times with PBST.

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

EXAMPLE 4, 3-3E efficacy against adenovirus 7 infection

In-vitro detection of 3-3E anti-human 7-type adenovirus infection prevention and treatment effects

1. A549 cells with good growth state are taken one day before experiment, are digested by pancreatin, and the cell density is adjusted to 3 multiplied by 10 percent by DMEM and 10 percent (volume percentage content) FBS⁵Perml, inoculated in 96-well cell culture plates, 100. mu.l per well, 5% CO at 37 ℃₂And (5) culturing.

2. After the step 1 is completed, taking out a 96-well plate on the same day of the experiment, removing the culture medium, washing the culture medium once without serum, and adding DMEM + 2% (volume percentage content) FBS into each well by 100 mu l; the packet then proceeds as follows:

experimental group (3-3E 0 h): diluting the 3-3E antibody prepared in example 2 with serum-free medium to obtain test antibody solutions containing 3-3E antibodies at different concentrations (50. mu.g/ml, 25. mu.g/ml, 12.5. mu.g/ml, 6.25. mu.g/ml, 3.2. mu.g/ml, 1.6. mu.g/ml, 0.8. mu.g/ml, 0.4. mu.g/ml, 0.2. mu.g/ml); the antibody solution to be tested was mixed with the HAdV7 virus stock solution prepared in step one 1 of example 3 (diluted with serum-free medium to a virus concentration of 2X 10)³TCID50/mL) were mixed at a volume ratio of 1:1, incubated at 37 ℃ for 1.5h, added to the wells, and incubated at 37 ℃ for 1h in a 5% CO2 incubator.

Experimental group (3-3E +1 h): the 3-3E antibody prepared in example 2 was diluted with a serum-free medium to obtain test antibody solutions containing 3-3E antibodies at different concentrations ((50. mu.g/ml, 25. mu.g/ml, 12.5. mu.g/ml, 6.25. mu.g/ml, 3.2. mu.g/ml, 1.6. mu.g/ml, 0.8. mu.g/ml, 0.4. mu.g/ml, 0.2. mu.g/ml), and the HAdV7 virus stock solution prepared in step one of example 3 (diluted with a serum-free medium to a virus concentration of 2X 10)³TCID50/mL) was added to the wells and incubated at 37 ℃ for 1h, then an antibody solution to be tested, which had an equal volume to the virus solution, was added to the wells and incubated at 37 ℃ for 1h in a 5% CO2 incubator.

Irrelevant antibody control group (Anti-DENV 1): an Anti-DENV1 Antibody (described in the literature: patent conjugation stability of a Human Monoclonal Antibody Against agar section 1Dengue Virus. frontiers in Microbiology, 1June2018, Volume 9, Article 1214; publicly available from the military medical research institute of the national military academy of liberation military sciences) was diluted with serum-free medium to obtain test Antibody solutions containing different concentrations (25. mu.g/ml, 12.5. mu.g/ml, 6.25. mu.g/ml, 3.2. mu.g/ml, 1.6. mu.g/ml, 0.8. mu.g/ml, 0.4. mu.g/ml, 0.2. mu.g/ml and 0.1. mu.g/ml) of Anti-DG Antibody; the antibody solution to be tested was mixed with the HAdV7 virus stock solution prepared in step one 1 of example 3 (diluted with serum-free medium to a virus concentration of 2X 10)³TCID50/mL) were mixed at a volume ratio of 1:1, incubated at 37 ℃ for 1.5h, added to the wells, and incubated at 37 ℃ for 1h in a 5% CO2 incubator.

Irrelevant antibody control group (Anti-EGFR): diluting Anti-EGFR antibody (described in patent CN102993305B) with serum-free culture medium to obtain test antibody solutions containing Anti-DG antibodies with different concentrations (25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.2 μ g/ml, 1.6 μ g/ml, 0.8 μ g/ml, 0.4 μ g/ml, 0.2 μ g/ml and 0.1 μ g/ml); the antibody solution to be tested was mixed with the HAdV7 virus stock solution prepared in step one 1 of example 3 (diluted with serum-free medium to a virus concentration of 2X 10)³TCID50/mL) were mixed at a volume ratio of 1:1, incubated at 37 ℃ for 1.5h, added to the wells, and incubated at 37 ℃ for 1h in a 5% CO2 incubator.

Virome (VIRUS): the HAdV7 virus stock solution prepared in step one of example 3 was diluted with serum-free medium to a virus concentration of 2X 10³TCID50/mL) at a volume ratio of 1:1, incubating at 37 deg.C for 1.5h, adding to the well, adding 100 μ l/well, continuing to stand at 37 deg.C and 5% CO₂Incubate for 1 h.

CELL free Control (CELL): serum-free medium, incubated at 37 ℃ for 1.5h, added to the wells, and incubated at 37 ℃ for 1h in a 5% CO2 incubator.

3. After step 2, taking a 96-well plate, removing the supernatant, adding DMEM + 2% (volume percentage content) FBS culture medium (100 mu l/well), and continuously culturing for 1 week at 37 ℃ with 5% CO2 until the cells have obvious lesions; the plates were removed and observed under the mirror, and the number of wells of non-diseased, partially diseased, and fully diseased cells was counted to calculate the inhibition rate of the antibody against adenovirus infection (table 1).

TABLE 13-3E results of detection of anti-HAdV 7 infected cells

Concentration unit: mu g/mL; 0h, inoculating cells after the antibody and the virus are incubated together; +1 h: antibody supplementation 1 hour after viral infection of cells

The results show that the antibody 3-3E prepared by the invention can effectively inhibit the proliferation of HAdV7, and the EC50 is 0.4 mu g/mL when used for prevention, and the EC50 is 0.72 mu g/mL when used after infecting virus.

II, 3-3E anti-human 7 type adenovirus infected CB-17SCID mice

1. Establishment of animal model

CB-17SCID mice (Wittingle), female, 16-18g, weighed on the day of the experiment, randomly divided into experimental and control groups (12 per group), were anesthetized with isoflurane inhalation, and the experimental groups were nasally inoculated with the HAdV7 virus concentrate (10) prepared in step one, step 2, example 3⁸TCID 50/mouse), control group was nasally inoculated with an equal volume of medium. Body weight was measured daily and the test animals were observed for loss of hair, food intake, water intake and activity.

The results are shown in FIG. 6. The results show that the mice in the experimental group start to eat and drink less water the next day after being inoculated with the virus, the hairs become rough, the bodies are thinned, the symptoms last about 5 days, the bodies of the control groups do not obviously change, the body weights continuously increase, and the fact that the mice in the experimental group are infected with the ADV7 virus is confirmed.

2. Effect of antibody 3-3E antibodies on uninfected mice

CB-17SCID mice (Viton Rivay), female, 16-18g are taken, weighed on the day of the experiment, randomly divided into an experiment group and a control group (12 mice in each group), isoflurane is inhaled for anesthesia, the 3-3E antibody prepared in example 2 is diluted by serum-free DMEM medium to obtain an antibody solution containing 200mg/ml of the 3-3E antibody, the nose of each experiment group is dripped into 20 mu l, and the control group is inoculated with the same volume of the serum-free medium. Body weight was measured daily and the test animals were observed for loss of hair, food intake, water intake and activity.

The results are shown in FIG. 7. The results show that the bodies of the experimental groups and the control groups have no obvious change and the body weight is continuously increased, and the experimental group antibody inoculation can not have obvious influence on the mice.

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

CB-17SCID mice (Witongliwa), female, 16-18g, were weighed on the day of the experiment and randomly assigned to experimental and control groups (12 per group) as follows:

experimental group (3-3E): diluting the prepared 3-3E antibody with serum-free DMEM medium to obtain an antibody solution containing 200ug/ml 3-3E antibody; the antibody solution was mixed with the antibody solution obtained in step one of example 32 (virus concentration 10) of the HAdV7 virus concentrate¹⁰TCID50/ml) were mixed at a volume ratio of 1:1, mixed and incubated at 37 ℃ for 1h, the experimental group of mice prepared in step 1 was inoculated nasally, 20 μ l each, and the mice were observed for depilation, feeding, drinking and mobility and the body weight was recorded.

Control group (VIRUS-only): the concentrated solution of HAdV7 virus prepared in step one 2 of example 3 (virus concentration 10)¹⁰TCID50/ml) was mixed with serum-free DMEM medium at a volume ratio of 1:1, incubated at 37 ℃ for 1 hour, the experimental group mice prepared in step 1 (each inoculated with 20 μ l) were inoculated nasally, and the mice were observed for depilation, feeding, drinking and movement and the body weight thereof was recorded.

The results are shown in FIG. 8. The results show that the control group shows new and obvious symptoms of body form emaciation, hair color roughness and the like in the next day, and the symptoms of the experimental group mouse are obviously relieved compared with the control group, which shows that the 3-3E antibody can effectively relieve the symptoms of weight loss, hair color roughness and the like of the mouse caused by virus infection.

Sequence listing

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Claims

1. A monoclonal antibody against human adenovirus 7, 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 HCDR 3; the light chain variable region comprises three complementarity determining regions, LCDR1, LCDR2, and LCDR 3;

the amino acid sequence of the HCDR1 is shown as 26 th to 33 th sites from the N end of a sequence 2in a sequence table;

the amino acid sequence of the HCDR2 is shown as 51-58 th from the N end of the sequence 2in the sequence table;

the amino acid sequence of the HCDR3 is shown as 97 th-110 th site from the N end of a sequence 2in a sequence table;

the amino acid sequence of the LCDR1 is shown as 27 th to 32 th sites from the N end of a sequence 4 in a sequence table;

the amino acid sequence of the LCDR2 is shown as the 50 th-52 th site from the N end of the sequence 4 in the sequence table;

the amino acid sequence of the LCDR3 is shown as 89-96 th site from the N end of the sequence 4 of the sequence table.

2. The monoclonal antibody against human adenovirus 7 according to claim 1, wherein:

the amino acid sequence of the heavy chain variable region is shown as sequence 2in the sequence table;

the amino acid sequence of the light chain variable region is shown as a sequence 4 in a sequence table.

3. A gene encoding the monoclonal antibody against human adenovirus 7 according to claim 1 or 2.

4. The gene of claim 3, wherein:

the gene of the heavy chain variable region of the coded antibody is a DNA molecule shown in a sequence 1 of a sequence table;

the gene of the light chain variable region of the coded antibody is a DNA molecule shown in a sequence 3 of a sequence table.

5. Use of the monoclonal antibody against human adenovirus 7 according to claim 1 or 2 for the preparation of a medicament for the prevention and/or treatment of diseases caused by human adenovirus 7 infection.

6. Use of the monoclonal antibody against human adenovirus 7 according to claim 1 or 2 for the preparation of a product; the application of the product is (e1) and/or (e 2):

(e1) inhibiting human adenovirus type 7;

(e2) neutralizing human adenovirus type 7.

7. A medicament for preventing and/or treating diseases caused by human adenovirus 7 infection, which comprises the monoclonal antibody against human adenovirus 7 according to claim 1 or 2 as an active ingredient.

8. A product, the active ingredient of which is the monoclonal antibody against human adenovirus 7 according to claim 1 or 2; the application of the product is (e1) and/or (e 2):

(e1) inhibiting human adenovirus type 7;

(e2) neutralizing human adenovirus type 7.