CN109957009B

CN109957009B - Anti-human 7-type adenovirus antibody 2-1H and application thereof

Info

Publication number: CN109957009B
Application number: CN201910242175.6A
Authority: CN
Inventors: 杨志新; 王荣; 陆健昇; 余云舟; 周权; 黄莺; 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: 2021-03-19
Anticipated expiration: 2039-03-28
Also published as: CN109957009A

Abstract

The invention discloses an anti-human adenovirus 7 antibody 2-1H 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 117 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 97 th from the N end of a sequence 4 of a sequence table. Based on clinical requirements, the inventor discovers an anti-human adenovirus 7 antibody 2-1H, which is used for preventing and treating adenovirus infection and has important biological and medical significance.

Description

Anti-human 7-type adenovirus antibody 2-1H and application thereof

Technical Field

The invention relates to an anti-human adenovirus 7 antibody 2-1H 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 70-90 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 the adenovirus capable of causing respiratory tract infection has 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, IgG is generated later, most of IgM begins to be generated at 7-10 days, then 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, 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 antibody of 12 and 5 types of adenovirus, 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 treatment means, and the development of the adenovirus neutralizing antibody should be a main development direction of future adenovirus infection treatment.

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 an anti-human adenovirus 7 antibody 2-1H 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 amino acid sequence of 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 amino acid sequence of the HCDR3 is (a7) or (a8) or (a9) as follows:

(a7) an amino acid sequence shown in 97 th-117 th site from 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 of an amino acid sequence shown from 97 th to 117 th sites of the N end of the sequence 2in the sequence table;

(a9) an amino acid sequence which has homology of 75 percent or more than 75 percent with the amino acid sequence shown by 97 th to 117 th sites from the N end of the sequence 2in 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 97 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 97 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 percent or more than 75 percent with the amino acid sequence shown in the 89 th to 97 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 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 any one of the monoclonal antibodies.

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 an anti-human adenovirus 7 antibody 2-1H, which is used for preventing and treating adenovirus infection and has important biological and medical significance.

Drawings

FIG. 1 shows the expression and purification of 2-1H.

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 2-1H.

FIG. 5 is an affinity assay for 2-1H.

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

FIG. 7 is a graph of the protective effect of 2-1H 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 convalescent human 7 type adenovirus infected person, 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 2-1H antibody.

The amino acid sequence of the heavy chain variable region of the 2-1H antibody is shown as the sequence 2in the sequence table (wherein, the 26 th to 33 th amino acid residues from the N end form CDR1, the 51 th to 58 th amino acid residues form CDR2, and the 97 th to 117 th amino acid residues form CDR3), and the coding gene thereof is shown as the sequence 1 in the sequence table.

The amino acid sequence of the light chain variable region of the 2-1H antibody is shown as the sequence 4 in the sequence table (wherein, the 27 th to 32 th amino acid residues from the N end form CDR1, the 50 th to 52 th amino acid residues form CDR2, and the 89 th to 97 th amino acid residues form CDR3), and the coding gene thereof is shown as the sequence 3 in the sequence table.

EXAMPLE 2 preparation of 2-1H antibody

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, 2-1H antibody

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: diluting 24 μ l FectoPRO transfection reagent in 3ml FreeStyle 293 medium (Gibco12338-018), gently mixing, adding 12 μ g of the recombinant expression vector containing the heavy chain variable region prepared in step one and 12 μ g of the recombinant expression vector containing the light chain variable region, mixing, and standing 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, aktaexplor), 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 protein, 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 detected protein concentration was 1.05 mg/mL.

Example 3 detection of binding Capacity of 2-1H antibody

Preparation of human 7 type adenovirus stock solution, 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% CO₂Continuously culturing in the incubator; the cytopathic condition was observed daily (after proliferation of viral infection)Cytopathic effect, which is 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 digestion⁵The cells were inoculated in 96-well cell culture plates, 100. mu.l per well, cultured at 37 ℃ with 5% CO 2; 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 detected by 10 times of gradient of serum-free medium, 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

Transfer of virus stock solutionMoving to a 500ml sample bottle, adjusting the pH value to 7.6 by using sodium bicarbonate, then adding beta-propiolactone according to the proportion of 1:2000 while stirring, fully and uniformly mixing, continuously stirring and inactivating at 4 ℃, adjusting the pH value to 7.6 again after 24 hours, supplementing beta-propiolactone according to the proportion of 1:2000, continuously stirring and inactivating for 24 hours at 4 ℃, taking a sample with the volume of not less than 1 per thousand for hydrolysis in 37 ℃ water bath for 4 hours (adjusting the pH value to about 7.0 by using sodium bicarbonate when the color of the sample is observed to turn yellow in the middle), finishing the 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 (pH7.9-8) ] 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 (pH7.9-8) ] 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, 2-1H 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 2-1H 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 HAdV7 virus with influenza virus A/swine/Colorado/1/77(H3N2) according to the method of the step one) and a synthesized foot-and-mouth disease virus polypeptide antigen (sequence, ETQVQRRQHTDVSFILDRFVKVTPKDQINALDLMQTPAHTEPGSRVTNVRGDLQVLAQKAARTLPPGSRHKQKIVAPVKQLL) as antigens to replace the HAdV7 inactivated virus.

The results are shown in FIG. 4. The results indicate that the 2-1H antibody can specifically bind to inactivated adenovirus and not to influenza virus (Flu) or foot and mouth disease virus polypeptide antigen (FMDV).

Analysis of antigen binding Capacity of Tris, 2-1H 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 2-1H antibody solution (the initial concentration is 100 microgram/ml) diluted according to 2 times of equal ratio is added into each hole, 28 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 2-1H antibody to human adenovirus type 7 was EC50 ═ 0.036 nM.

Example 4, 2-1H efficacy against adenovirus 7 infection

In-vitro detection of prevention effect of 2-1H against human 7-type adenovirus infection

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 (2-1H): diluting the 2-1H antibody prepared in example 2 with serum-free medium to obtain test antibody solutions containing 2-1H 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) 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.

Irrelevant antibody control group (Anti-DENV 1): diluting Anti-DENV1 Antibody (described in patent publication: patent conjugation stability of a Human Monoclonal Antibody Against agar sections 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) with serum-free medium to obtain a test Antibody solution containing 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) of Anti-DG Antibody; the antibody solution to be tested was mixed with the HAdV7 virus stock solution (diluted with serum-free medium to a virus concentration of 2X 10) prepared in step one of example 3 in step 1³TCID50/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.

Irrelevant antibody control group (Anti-EGF): diluting Anti-EGFR antibody (described in patent CN102993305B) with serum-free culture medium to obtain test antibody solutions containing Anti-EGFR antibodies with different concentrations (50 μ g/ml, 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); 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)³CID50/mL) at a volume ratio of 1:1, incubating at 37 deg.C for 1.5h, adding into the well, adding 100 μ l/well, further placing 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 at 100. mu.l/well, 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.

3. After completing step 2, a 96-well plate was taken, the supernatant was discarded, and DMEM + 2% (volume percent) FBS medium (100. mu.l/well) was added thereto at 37 ℃ with 5% CO₂Continuing to culture for 1 week until the cells have obvious lesions; the plates were removed and observed under the microscope, and the number of wells with no lesions, partial lesions and complete lesions was counted, and the inhibition rate of the antibody against adenovirus infection was calculated using Graphpad Prism software (table 1).

TABLE 12-1H 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;

the result shows that the antibody 2-1H prepared by the invention can effectively inhibit the proliferation of HAdV7, and EC50 is 1.6 mu g/mL.

II, 2-1H anti-human 7 type adenovirus infected CB-17SCID mouse

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 inoculated nasally with equal volume of cultureAnd (4) a base. 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 are inoculated with the virus, the food intake and the water drinking are reduced at the next day, the hair is rough, the body is thin, the symptoms last about 5 days, the body of the control group is not obviously changed, the body weight continuously increases, and the fact that the mice in the experimental group are infected with the HAdV7 virus is proved.

2. 2-1H anti-human 7 type adenovirus infected CB-17SCID mouse

The following operations were performed for the experimental group and the control group:

CB-17SCID mice (Witongliwa), female, 16-18g, were weighed on the day of the experiment, randomized into experimental and control groups (12 per group), and subjected to isoflurane inhalation anesthesia.

Experimental group (2-1H): diluting the 2-1H antibody prepared in example 2 with serum-free DMEM medium to obtain an antibody solution containing 200ug/ml 2-1H antibody; the antibody solution was mixed with the HAdV7 virus concentrate (virus concentration 10) prepared in step one, step 2, of example 3¹⁰TCID50/ml) were mixed at a volume ratio of 1:1, incubated at 37 ℃ for 1h, nasally inoculated into CB-17SCID mice, 20 μ l each, and the mice were observed for loss of hair, feeding, drinking and mobility and recorded for body weight.

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 1h, nasally inoculated into CB-17SCID mice (each inoculated at 20 μ l), observed for loss of hair, feeding, drinking and mobility and recorded for weight.

The results are shown in FIG. 7. The results show that the control group shows new and obvious symptoms of body form emaciation, rough hair color and the like the next day, and the symptoms of the experimental group mouse are obviously lighter than those of the control group, which shows that the 2-1H antibody of the mouse can effectively reduce the symptoms of weight loss, rough hair color and the like of the mouse caused by virus infection.

Sequence listing

<110> military medical research institute of military science institute of people's liberation force of China

<120> anti-human adenovirus 7 antibody 2-1H and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 375

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cgggggggtc ccggagactc 60

tcctgtgcag cctctggatt caactttagg gactactcca tgacctgggt ccgccagact 120

ccagggaggg ggctggagtg gctctgtagt attagtgggg ttggtggcag tacacactac 180

gcagactccg tgaagggacg gttcaccatc tccagagaca attccaaaaa cacggtgtat 240

atgcaaatga acagcctgag agccgaggac acggccttat attactgcgc gagataccct 300

tatgatcatg tttgggggac ttttcgttat acgggcgcct ggtttgaccc ctggggccaa 360

gggaccacgg tcacc 375

<210> 2

<211> 125

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Ser Arg Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Arg Asp Tyr

20 25 30

Ser Met Thr Trp Val Arg Gln Thr Pro Gly Arg Gly Leu Glu Trp Leu

35 40 45

Cys Ser Ile Ser Gly Val Gly Gly Ser Thr His Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Tyr Pro Tyr Asp His Val Trp Gly Thr Phe Arg Tyr Thr Gly

100 105 110

Ala Trp Phe Asp Pro Trp Gly Gln Gly Thr Thr Val Thr

115 120 125

<210> 3

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gacatccaga tgacccagtc tccatattcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca ggatattagc agctggttag cctggtatca gcagaaacca 120

gggaaagccc ctaaactcct gatcaacgct gcatccactt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatt tgggacagat ttcactctca ccatcagcag cctgcagcct 240

gaagattttg caacctacta ttgtcagcag gctaacagtc tcccgtacac ttttggccaa 300

gggaccaagg tggaaatcaa acgaact 327

<210> 4

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Asn Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

Claims

1. A monoclonal antibody that binds human adenovirus type 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-117 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 th-97 th site from the N end of the sequence 4 of the sequence table.

2. The monoclonal antibody of claim 1, wherein:

the amino acid sequence of the heavy chain variable region is shown as a sequence 2in a 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 of 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 a monoclonal antibody according to claim 1 or 2 for the preparation of a medicament for the prevention and/or treatment of a disease caused by an adenovirus type 7 infection in a human.

6. Use of the monoclonal antibody of claim 1 or 2in the manufacture 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 a disease caused by adenovirus infection, which comprises the monoclonal antibody according to claim 1 or 2 as an active ingredient.

8. A product whose active ingredient is the monoclonal antibody of claim 1 or 2; the application of the product is (e1) and/or (e 2):

(e1) inhibiting adenovirus;

(e2) and (3) neutralizing the adenovirus.