CN109897104A - 7 type adenovirus monoclonal antibody 3-3E of people and its application - Google Patents
7 type adenovirus monoclonal antibody 3-3E of people and its application Download PDFInfo
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Abstract
The invention discloses a kind of 7 type adenovirus monoclonal antibody 3-3E of people and its applications.The present invention provides a kind of monoclonal antibodies, including heavy chain variable region and light chain variable region；The heavy chain variable region includes three complementary determining regions HCDR1, HCDR2 and HCDR3；The light chain variable region includes three complementary determining regions LCDR1, LCDR2 and LCDR3；Described HCDR1, HCDR2 and HCDR3 are successively if the sequence of sequence table 2 is from shown in N-terminal 26-33,51-58,97-110；Described LCDR1, LCDR2 and LCDR3 are successively if the sequence of sequence table 4 is from shown in N-terminal 27-32,50-52,89-96.The present inventor is based on clinical demand, it was found that anti-human 7 type adenovirus antibody 3-3E has important biology and medical significance for the prevention and treatment of adenovirus infection.
Description
Technical field
The present invention relates to a kind of 7 type adenovirus monoclonal antibody 3-3E of people and its applications.
Background technique
Adenovirus hominis belongs to Adenoviridae mastadenovirus, and adenovirus hominis is found in nineteen fifty-three, separation training earliest
Support the almond adenoid tissue from Healthy People atrophy；The genome of virus is distrand DNA, overall length about 36kb, these double-stranded DNAs and disease
Malicious structural proteins combine composition virus core, and virus is coated with capsid without coating, core outside, and the capsid is by 252 capsomere groups
At, wherein 240 be hexon, 12 penton proteins, shell in rule 20 face body structures, diameter about 80~
110nm。
The adenovirus having now been found that has A-G totally 7 subgroups, 67 different serotypes, wherein can infect people and cause a disease
Have 55 hypotypes.Adenovirus infection people most commonly infects respiratory tract and causes respiratory disease, furthermore fractionated viral
It can also cause the system infections such as uropoiesis and stomach and intestine, research shows that causing the adenovirus of respiratory tract infection mainly has A, C, E and B1
Group, wherein mainly B1 subgroup, B2 subgroup can infect the urinary system of people, and F, D group can infect the gastronintestinal system and knot of people respectively
Membranous system.It is currently known the type of cause the adenovirus of respiratory tract infection to have B1 subgroup in the world 1,2,3,4,7,14 and 55, wherein 3
Type, 4 types, 7 types, 14 types be the most common type for causing epidemic outbreaks, the China Zeng Yinqi Jiangsu and Taiwan Province and South Korea,
The Respiratory Tract Adenovirus epidemic outbreaks such as Singapore, Malaysia.Since two thousand eight, a lot of warps successively occur for China different regions
Respiratory infectious breaks out epidemic, and epidemic situation involves wide, and infectiousness is strong, is respectively B through the identification of laboratory Pathogen test
7 types of group, 55 types and 14 type adenovirus.
Under normal circumstances, when virus-infected human, human immune system can excite humoral immunity and cell immune response
And gradually control infection, removing virus, 3 days or so after the onset of infecting human body and causing, the IgM of patient's body is opened most of virus
Begin to generate, IgG generates major part a little later and starts to generate in 7~10d, then gradually rises, reaches peak within general 1 month or so.
Therefore the important role of performer, adenovirus infection human body can also induce stronger neutralizing antibody always in virus infection rehabilitation
Humoral immune reaction generates specific antibody.Research shows that body to homotype adenovirus again subinfection can produce it is effectively immune, and
The immunoprotection (up to 10 years or more) that can produce the long period, after rehabilitation generally will not subinfection again, and wherein work
The neutralizing antibody that be exactly adenovirus infection process generate at induction body, find the 6~15 of China 40%~60% according to the study
The people in year has 1,2 and 5 type neutralizing antibodies, and the antibody of mother can protect baby to exempt serious adenovirus infection.Therefore
Adenovirus neutralizing antibody is considered as that can be used as a kind of effectively special anti-Adenoviral Therapy means, adenovirus neutralizing antibody
Development should be the exploitation of following adenovirus infection therapeutic agent a main direction of development.
At present in the world still without for adenovirus specific treatment drug for clinic, medicament research and development also more being limited to
The nucleoside analog of medicine --- resisting DNA virus, and for the research of the specific biological medicine of adenovirus or blank.
Summary of the invention
The object of the present invention is to provide a kind of 7 type adenovirus monoclonal antibody 3-3E of people and its applications.
Present invention firstly provides a kind of monoclonal antibodies, including heavy chain variable region and light chain variable region；The heavy chain can
Becoming area includes three complementary determining regions HCDR1, HCDR2 and HCDR3；The light chain variable region includes three complementary determining regions
LCDR1, LCDR2 and LCDR3；
The amino acid sequence of the HCDR1 is following (a1) or (a2) or (a3):
(a1) sequence 2 of sequence table amino acid sequence shown in N-terminal 26-33；
(a2) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 26-33 passes through one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of residue obtain；
(a3) have 75% or 75% or more with the sequence 2 of sequence table amino acid sequence shown in N-terminal 26-33
Homology and amino acid sequence with the same function；
The HCDR2 is following (a4) or (a5) or (a6):
(a4) sequence 2 of sequence table amino acid sequence shown in N-terminal 51-58；
(a5) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 51-58 passes through one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of residue obtain；
(a6) have 75% or 75% or more with the sequence 2 of sequence table amino acid sequence shown in N-terminal 51-58
Homology and amino acid sequence with the same function；
The HCDR3 is following (a7) or (a8) or (a9):
(a7) sequence 2 of sequence table amino acid sequence shown in N-terminal 97-110；
(a8) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 97-110 passes through one or several amino
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of sour residue obtain；
(a9) with the sequence 2 of sequence table amino acid sequence shown in N-terminal 97-110 with 75% or 75% or more
Homology and amino acid sequence with the same function；
The amino acid sequence of the LCDR1 is following (b1) or (b2) or (b3):
(b1) sequence 4 of sequence table amino acid sequence shown in N-terminal 27-32；
(b2) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 27-32 passes through one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of residue obtain；
(b3) have 75% or 75% or more with the sequence 4 of sequence table amino acid sequence shown in N-terminal 27-32
Homology and amino acid sequence with the same function；
The amino acid sequence of the LCDR2 is following (b4) or (b5) or (b6):
(b4) sequence 4 of sequence table amino acid sequence shown in N-terminal 50-52；
(b5) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 50-52 passes through one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of residue obtain；
(b6) have 75% or 75% or more with the sequence 4 of sequence table amino acid sequence shown in N-terminal 50-52
Homology and amino acid sequence with the same function；
The amino acid sequence of the LCDR3 is following (b7) or (b8) or (b9):
(b7) sequence 4 of sequence table amino acid sequence shown in N-terminal 89-96；
(b8) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 89-96 passes through one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of residue obtain；
(b9) have 75% or 75% or more with the sequence 4 of sequence table amino acid sequence shown in N-terminal 89-96
Homology and amino acid sequence with the same function.
The amino acid sequence of the heavy chain variable region is following (c1) or (c2) or (c3):
(c1) amino acid sequence shown in the sequence 2 of sequence table；
(c2) by amino acid sequence shown in the sequence of sequence table 2 by one or several amino acid residues substitution and/
Or the amino acid sequence with the same function that deletion and/or addition obtains；
(c3) homology with amino acid sequence shown in the sequence of sequence table 2 with 75% or 75% or more and have phase
The amino acid sequence of congenerous；
The amino acid sequence of the light chain variable region is following (c4) or (c5) or (c6):
(c4) amino acid sequence shown in the sequence 4 of sequence table；
(c5) by amino acid sequence shown in the sequence of sequence table 4 by one or several amino acid residues substitution and/
Or the amino acid sequence with the same function that deletion and/or addition obtains；
(c6) homology with amino acid sequence shown in the sequence of sequence table 4 with 75% or 75% or more and have phase
The amino acid sequence of congenerous.
The present invention also protects the gene of encoding said antibody.
The gene of the heavy chain variable region of encoding said antibody is following (d1) or (d2) or (d3):
(d1) DNA molecular shown in the sequence 1 of sequence table；
(d2) nucleotide sequence limited with (d1) has 75% or 75% or more homology, and encodes claims 1 or 2
Described in heavy chain variable region DNA molecular；
(d3) nucleotide sequence hybridization limited under strict conditions with (d1) or (d2), and encode in claims 1 or 2
The DNA molecular of the heavy chain variable region；
The gene of the light chain variable region of encoding said antibody is following (d4) or (d5) or (d6):
(d4) DNA molecular shown in the sequence 3 of sequence table；
(d5) nucleotide sequence limited with (d4) has 75% or 75% or more homology, and encodes claims 1 or 2
Described in heavy chain variable region DNA molecular；
(d6) nucleotide sequence hybridization limited under strict conditions with (d4) or (d5), and encode in claims 1 or 2
The DNA molecular of the heavy chain variable region.
The present invention also protects any description above monoclonal antibody to draw in preparation for preventing and/or treating adenovirus infection
Application in the drug of the disease risen.
The present invention also protects any description above monoclonal antibody preparing the application in product；The purposes of the product is
As follows (e1) and/or (e2):
(e1) inhibit adenovirus；
(e2) adenovirus is neutralized.
The present invention also protect it is a kind of for preventing and/or treating the drug of disease caused by adenovirus infection, activity at
It is divided into the monoclonal antibody of any description above.
The present invention also protects a kind of product, and active constituent is the monoclonal antibody of any description above；The product
Purposes is following (e1) and/or (e2):
(e1) inhibit adenovirus；
(e2) adenovirus is neutralized.
Any description above adenovirus is adenovirus hominis.7 type adenovirus of the adenovirus hominis behaviour.
The present inventor is based on clinical demand, it was found that anti-human 7 type adenovirus antibody 3-3E is used for adenovirus infection
Prevention and treatment, have important biology and medical significance.
Detailed description of the invention
The expression and purification that Fig. 1 is 3-3E detects.
Fig. 2 is the SDS-PAGE electrophoresis detection of HAdV7.
The Electronic Speculum that Fig. 3 is HAdV7 detects.
Fig. 4 is the antigenic specification analysis of 3-3E.
Fig. 5 is the affinity analysis of 3-3E.
Fig. 6 is the building of adenovirus infection animal model.
Fig. 7 is impact analysis of the 3-3E inoculation to CB-17SCID mouse weight.
Fig. 8 is the protective effect that 3-3E infects HAdV7 CB-17SCID mouse.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
HAdV7 virus: human/CHN/GZ6965/2001 is recorded in document: Yu Z, Zeng Z, Zhang J, et
al.Fatal Community-acquired Pneumonia in Children Caused by Re-emergent Human
Adenovirus 7d Associated with Higher Severity of Illness and Fatality Rate
[J].Scientific Reports,2016,6:37216.；The public can cure from PLA Academy of Military Sciences's military affairs
Research institute is learned to obtain.
Influenza antigen: A/swine/Colorado/1/77 (H3N2) is recorded in document: KarasinAI,
Schutten M M,Cooper LA,et al.Genetic characterization ofH3N2influenza viruses
isolated from pigs in North America,1977-1999:Evidence for wholly human and
reassortant virus genotypes[J].Virus Research,2000,68(1):71-85.；The public can be therefrom
Military medical research institute, PLA Academy of Military Sciences, state obtains.
The discovery of embodiment 1, antibody
Convalescence DENV-1 the infected peripheral blood is acquired, therefrom separates and collects peripheral blood mononuclear lymphocyte.It collects
Corresponding molecular labeling antibody (BD, #555332, #555415, #555441, the # in anti-cell surface is added in peripheral blood mononuclear lymphocyte
560677, #555622) or control antibodies (BD, #555748, #555742, #555751, #555749, #557872) are marked
Note, then carries out airflow classification, and the cell of sorting is expanded for antibody gene.Using the single B cell of separation as template, amplification is anti-
Body gene using 7 type Adenovirus Purification samples as antigen, by largely screening, divides the positive cloning and sequencing of amplification and expression
Analysis, verifying, obtain 1 antibody sequence, are named as 3-3E antibody.
The amino acid sequence of the heavy chain variable region of 3-3E antibody is as shown in the sequence 2 of sequence table (wherein, from N-terminal 26-33
Amino acids residue forms CDR1, and 51-58 amino acids residue forms CDR2,97-110 amino acids residue composition
CDR3), encoding gene is as shown in the sequence 1 of sequence table.
The amino acid sequence of the light chain variable region of 3-3E antibody is as shown in the sequence 4 of sequence table (wherein, from N-terminal 27-32
Amino acids residue forms CDR1, and 50-52 amino acids residue forms CDR2,89-96 amino acids residue composition
CDR3), encoding gene is as shown in the sequence 3 of sequence table.
The preparation of embodiment 2,3-3E antibody
One, the building of recombinant plasmid
1, by the small fragment between pTSE-G1n carrier (hundred Te Meibo biology Co., Ltd of Beijing) site SalI and PmlI
DNA molecular shown in the sequence 1 of sequence table is replaced with, the recombinant expression carrier containing heavy chain variable region is obtained and (has been sequenced and has tested
Card).
2, the small fragment between pTSE-K carrier (hundred Te Meibo biology Co., Ltd of Beijing) site SalI and PmlI is replaced
It is changed to DNA molecular shown in the sequence 3 of sequence table, the recombinant expression carrier containing light chain variable region is obtained and (has been sequenced and has tested
Card).
Two, the preparation of 3-3E antibody
1, the day before transfection is by FreeStyleTMHEK 293-F cell (Invitrogen company, article No.: R79007) is adjusted
It is whole to concentration be 1.0 × 106/ ml, is seeded in culture bottle, 37 DEG C, 5%CO2, shake culture is for 24 hours in 125rpm cell shaking table.
2, after completing step 1, on the transfection same day, the culture bottle is taken, transfection composite is added in culture bottle, 37 DEG C, 5%
CO2, shake culture in 125rpm cell shaking table, start within 48 hours to monitor cell viability, when cell viability drops to 80-85% 1,
000rpm is centrifuged 10min and collects culture supernatant, and SDS-PAGE electrophoresis detection determines antibody expression (swimming lane 2 of Fig. 1).
Transfection composite: the FectoPRO transfection reagent of 24 μ l is diluted in 293 culture medium of 3ml FreeStyle
(Gibco 12338-018), mixes gently, and the 12 μ g of recombinant expression carrier containing heavy chain variable region of step 1 preparation is added,
12 μ g of recombinant expression carrier containing light chain variable region, is placed at room temperature for 10min after mixing.
3, after completing step 2, supernatant is removed into impurity with 0.45 μm of membrane filtration, 10 × PB is added and adjusts ion concentration
It is close with combination buffer；Antibody purification is carried out with AKTA purification system (GE, AKTA EXPLORER), purifies instrument in AKTA
Middle installation HiTrap MabSelect Xtra purification column, set corresponding system parameter, with combination buffer balance purification column and on
Sample continues thereafter with balance, then rinses prepacked column with citric acid solution (pH3.0) with antibody elution albumen, UV280 reaches 100
When start to collect, UV280 terminates to collect when being down to 100, and by buffer exchange be citrate solution (pH6.0).
4, the antibody-solutions for taking step 3 to purify, SDS-PAGE electrophoresis detection antibody expression (swimming lane 3 of Fig. 1), and sample use
NanoDrop ultraviolet specrophotometer (Thermo Scientific) measures protein concentration, is 1.25mg/ through detection protein concentration
mL。
Embodiment 3, the detection of the binding ability of 3-3E antibody
One, the preparation of 7 type adenovirus virus stock solution used, viral concentration liquid and inactivation of viruses of people
1, prepared by adenovirus virus liquid
Adenovirus culture culture: conventional DMEM+10% (volumn concentration) FBS culture medium culture A549 cell (Beijing
Article No.: 25) consonance cell resource center, passes on A549 cell in 75cm on the day before virus inoculation2In cell bottle, make second
Cell density reaches 75%~90% when its virus inoculation；The inoculation same day is slowly sucked out the cell culture medium in culture bottle, is added
5ml DMEM is discarded after gently rinsing cell, another that 3ml DMEM+2% (volumn concentration) FBS is added；It is inhaled with micropipettor
It takes appropriate HAdV7 virus in cell bottle, is infected by MOI ≈ 0.001, uniformly shake bottle makes virus dispersion equal for several times
It is even, 37 DEG C are placed in, 5%CO2Incubator in adsorb 2h, it is during which primary every 30min double swerve culture bottle；Absorption is completed
Afterwards, virus-culturing fluid is discarded, 15ml new DMEM+2% (volumn concentration) FBS is rejoined, then places cell bottle
At 37 DEG C, 5%CO2Incubator in continue to cultivate；Daily observation cytopathy situation (will appear cell after virus infection proliferation
Lesion such as shows as cell shrinkage, falls off at the features)；The harvest when lesion occurs in 75%-100% cell, -80 DEG C freeze thawing 2 times,
4,000rpm centrifugation 5min, are stored in -80 DEG C, as virus stock solution used after collecting supernatant packing.
2, the preparation of adenovirus concentrate: the viral cultures of harvest, 4,000rpm 10 minutes removal cell fragments of centrifugation,
Supernatant is transferred to the super filter tube (MILIPORE, article No. UFC805008) that molecular cut off is 50kD, and 4,000rpm are centrifuged to volume and subtract
As little as the 1/30 of initial volume collects trapped fluid, -80 DEG C, as viral concentration liquid is stored in after packing.
3, adenovirus titer determination
Experiment the previous day take the good A549 cell of growth conditions (Beijing coordinate cell resource center, article No.: 25), pancreatin
DMEM+10% (volumn concentration) FBS tune cell density is to 3 × 10 after digestion5/ ml is inoculated in 96 porocyte culture plates, often
100 μ l of hole, 37 DEG C, 5%CO2Culture；Experimental day takes out 96 orifice plates, abandons culture medium, and serum free medium is washed one time, is added
DMEM+2% (volumn concentration) FBS, 100 holes μ l/；Then 10 times of gradient dilution viruses to be measured of serum free medium are used
Liquid, 10-1~10-8Totally 8 gradients, the virus diluted are added in 96 orifice plates got ready according to 10 holes μ l/, each dilution gradient 8
Hole, while blank control group is set, operation completes cell and is placed in 37 DEG C, 5%CO2Incubator in cultivate observation, counted after 7 days
Each hole cell death situation calculates the TCID50 of virus stock solution used according to following formula.
Distance proportion=(higher than the percentage -50% of 50% lesion rate)/(percentage-higher than 50% lesion rate is lower than
The percentage of 50% lesion rate)
The logarithm of the dilution of 50% lesion rate of difference between LgTCID50=distance proportion × dilution logarithm+be higher than
After testing and calculation, in this research HAdV7 virus stock solution used used virus titer=5.0 × 108TCID50/ml,
Virus titer=1.0 × 10 of viral concentration liquid10TCID50/ml。
4, the inactivation and purifying of adenovirus
Virus stock solution used is taken to be transferred to 500ml sample bottle, with sodium bicarbonate tune pH to 7.6, then according to the ratio of 1:2000
Beta-propiolactone is added, stirring while adding, after mixing well, 4 DEG C are continued stirring inactivation, pH to 7.6 is adjusted again after 24 hours, according to
The ratio of 1:2000 adds beta-propiolactone, and 4 DEG C are continued stirring inactivation 24 hours, takes 37 DEG C of water-baths of sample of no less than 1 ‰ volumes
Hydrolysis 4 hours (centre, which is observed, adjusts pH to 7.0 or so with sodium bicarbonate when color sample turns yellow), hydrolysis terminated, and takes previous
It reaches 25cm2The A549 cell of cell bottle, according to 1 bottle of 25cm of 1ml sample inoculation2The ratio of A549 cell is inoculated with (less than 1ml
Calculated by 1ml), while the cell for being inoculated with non-inactivation of viruses is set and does not dispose ghost as control, it is placed in 37 DEG C, 5%CO2
Incubator in cultivate observation, blind passage continues to observe, such 3 generation of blind passage to new A549 cell after 7 days, and inoculation does not inactivate disease
Malicious sample cell lesion and inactivation experiments group cell and blanc cell not lesion person, it is credible to be judged to inactivating testing result, goes out
Living otherwise then inactivation is incomplete completely, needs to inactivate again, detect.
Detecting through inactivation confirms to inactivate thorough virus stock solution used, and 4,000rpm 10 minutes removal cell fragments of centrifugation use PBS
Buffer balances Sepharose 4Fast Flow gel column and loading, is then eluted with PBS, for the purpose of first eluting peak is
The eluting peak is collected at viral peak, and the CsCl of 12.5ml weight density is then added in Amicon-Ultra-15 super filter tube (50kD)
Solution [the 10mM Tris-HCL (PH 7.9-8) of 42.23g cesium chloride+57.77ml], is slow added into the light density of 12.5ml
CsCl solution [the 10mM Tris-HCL (PH 7.9-8) of 22.39g cesium chloride+77.61ml], the virus for adding 15ml suspend
Liquid, trim are put in ultracentrifuge (Beckman L100-XP), and 25,000rpm, 4 DEG C are centrifuged 2 hours.It collects in light density
Band between weight density cesium chloride solution, PBS dialysis, filtration sterilization obtain HAdV7 inactivation of viruses.
Sampling carries out routine SDS-PAGE detection and Electronic Speculum observation respectively.Electron microscopic sample preparation process is as follows: sampling 2.5%
Glutaraldehyde (0.075%PBS, pH7.4 dilution), 4 DEG C are fixed 2 hours；Then 15-20ul liquid to be checked is taken, it is (carbon containing to drop to copper mesh
Support film), it is incubated at room temperature 5-10min；Filter paper blots liquid；3% phosphotungstic acid (distilled water preparation) room temperature dyes 2min；Filter paper is inhaled
Dry liquids；Upper machine observation.
SDS-PAGE result is shown in Fig. 2, and Electronic Speculum result is shown in Fig. 3.
Two, 7 type inactivated adenovirus of 3-3E antibody specificity combination people
1, the HAdV7 inactivation of viruses (200ng) prepared in the 4 of 4 μ l step 1 is taken, carbonate is mended and is coated with buffer (pH
9.6) it to 100ul, is added in ELISA Plate (Corning 9018) by every hole 100ul, is arranged multiple holes 3,4 DEG C of coatings are overnight.
2, after completing above-mentioned steps, the ELISA Plate is taken, PBST board-washing 3 times, is added and contains 2% (mass percentage)
The PBS confining liquid of BSA, 37 DEG C are incubated for 2 hours.
3, after completing above-mentioned steps, the ELISA Plate is taken, discards confining liquid, the 3-3E antibody stoste of 100 μ l is added in every hole
(300 μ g/ml of concentration), 37 DEG C of incubation 90min, then PBST (PBS+0.1%Tween20) board-washing 3 times.
4, after completing above-mentioned steps, above-mentioned ELISA Plate is taken, 1:4000 times of 100 μ l of every hole addition dilutes enzyme labelled antibody (mountain
Goat anti-human igg-HRP, Zhong Shan Golden Bridge, article No. ZB-2304), 37 DEG C of incubation 45min, then PBST (PBS+0.1%Tween20)
Board-washing 3 times.
5, after completing above-mentioned steps, the ELISA Plate is taken, every hole is added 50 μ l OPD substrate developing solutions, is incubated for 10 at room temperature
Minute.
6, after completing above-mentioned steps, the ELISA Plate is taken, every hole is added 50 μ l 1M sulfuric acid solutions and terminates integrated enzyme reaction.
7, microplate reader 492nm/630nm dual wavelength measures OD value.
Above step is arranged simultaneously using inactivating influenza virus antigen (by influenza virus A/swine/Colorado/1/77
(H3N2) substitution HAdV7 virus, be prepared according to the method for step 1) as antigen substitute HAdV7 inactivation of viruses control
Group.
As a result as shown in Figure 4.The result shows that 3-3E antibody can specifically bind inactivated adenovirus without combining influenza disease
Malicious (Flu).
Three, the antigen binding capacity analysis of 3-3E antibody
1, the HAdV7 inactivation of viruses (200ng) prepared in the 4 of 4 μ l step 1 is taken, carbonate is mended and is coated with buffer (pH
9.6) to 100 μ l, being added to ELISA Plate by every hole 100ul, (Corning article No.: in 9018), setting multiple holes 3,4 DEG C were coated with
Night.
2, after completing above-mentioned steps, the ELISA Plate is taken, PBST board-washing 3 times, is added and contains 2% (mass percentage)
The PBS confining liquid of BSA, 37 DEG C are incubated for 2 hours.
3, after completing above-mentioned steps, the ELISA Plate is taken, discards confining liquid, 100 μ l are added according to 2 times of proportional diluteds in every hole
3-3E antibody liquid (400 μ g/ml of initial concentration), 30 gradients, 37 DEG C of incubation 90min, then PBST board-washing 3 are set altogether
It is secondary.
4, after completing above-mentioned steps, the ELISA Plate is taken, 1:4000 times of 100 μ l of every hole addition dilutes the anti-of HRP label
Human IgG antibody (Zhong Shan Golden Bridge, article No. ZB-2304), 37 DEG C of incubation 45min, PBST board-washing 3 times.
5, after completing above-mentioned steps, the ELISA Plate is taken, every hole is added 50 μ l OPD substrate developing solutions, is incubated for 10 at room temperature
Minute.
6, after completing above-mentioned steps, the ELISA Plate is taken, every hole is added 50 μ l 1M sulfuric acid solutions and terminates integrated enzyme reaction.
7, microplate reader 492nm/630nm dual wavelength measures OD value.
As a result as shown in Figure 5.In Fig. 5, abscissa is the logarithm of protein concentration, and ordinate is OD value.Analysis is aobvious
The binding ability for showing 3-3E antibody and 7 type adenovirus of people is EC50=339.3nM.
The drug effect of the anti-human 7 type adenovirus infection of embodiment 4,3-3E
One, the prevention and treatment of the anti-human 7 type adenovirus infection of vitro detection 3-3E
1, experiment the previous day takes the cell of the good A549 of growth conditions, and pancreatin digestion, (volume basis contains DMEM+10%
Amount) FBS tune cell density is to 3 × 105/ ml is inoculated in 96 porocyte culture plates, every hole 100 μ l, 37 DEG C of 5%CO2Culture.
2, after completing step 1, experimental day takes out 96 orifice plates, abandons culture medium, and serum free medium is washed one time, and DMEM is added
+ 2% (volumn concentration) FBS, every 100 μ l of hole；Then grouping proceeds as follows:
Experimental group (3-3E 0h): 3-3E antibody prepared by embodiment 2 is diluted with serum free medium, is obtained containing not
Same concentration (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) 3-3E antibody test antibodies solution；By what is prepared in test antibodies solution and the 1 of 3 step 1 of embodiment
(be diluted to virus concentration with serum free medium is 2 × 10 to HAdV7 virus stock solution used3TCID50/mL) mixed according to volume ratio 1:1
It closes, is added in hole after 37 DEG C of incubation 1.5h, continue to be placed in 37 DEG C, be incubated for 1h in the incubator of 5%CO2.
Experimental group (3-3E+1h): 3-3E antibody prepared by embodiment 2 is diluted with serum free medium, is obtained containing difference
Concentration ((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) 3-3E antibody test antibodies solution；The HAdV7 virus stock solution used prepared in the 1 of 3 step 1 of embodiment (is used
It is 2 × 10 that serum free medium, which is diluted to virus concentration,3TCID50/mL it) adds in hole, it, will be with virus liquid after 37 DEG C of incubation 1h
Isometric test antibodies solution adds in hole, continues to be placed in 37 DEG C, is incubated for 1h in the incubator of 5%CO2.
Irrelevant antibody control group (Anti-DENV1): Anti-DENV1 antibody (is recorded in document: Potent
Neutralization Ability of a Human Monoclonal Antibody Against Serotype
1Dengue Virus.Frontiers in Microbiology, 1June2018, Volume 9, Article 1214；The public
Can be obtained from military medical research institute, PLA Academy of Military Sciences) it is diluted with serum free medium, contained
Various concentration (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) Anti-DG antibody test antibodies solution；It will be made in test antibodies solution and the 1 of 3 step 1 of embodiment
(be diluted to virus concentration with serum free medium is 2 × 10 to standby HAdV7 virus stock solution used3TCID50/mL) according to volume ratio 1:1
It mixes, is added in hole after 37 DEG C of incubation 1.5h, continue to be placed in 37 DEG C, be incubated for 1h in the incubator of 5%CO2.
Irrelevant antibody control group (Anti-EGFR): Anti-EGFR antibody (is recorded in patent of invention patent
CN102993305B) diluted with serum free medium, obtain containing various concentration (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) Anti-DG antibody test antibodies
Solution；The HAdV7 virus stock solution used prepared in test antibodies solution and the 1 of 3 step 1 of embodiment (is diluted with serum free medium
It is 2 × 10 to virus concentration3TCID50/mL it) is mixed according to volume ratio 1:1, is added in hole after 37 DEG C of incubation 1.5h, continue to be placed in
37 DEG C, 1h is incubated in the incubator of 5%CO2.
Viral group (VIRUS): the HAdV7 virus stock solution used that will be prepared in serum free medium and the 1 of 3 step 1 of embodiment
(being diluted to virus concentration with serum free medium is 2 × 103TCID50/mL it) is mixed according to volume ratio 1:1,37 DEG C of incubation 1.5h
After add in hole, 100 holes μ l/ continue to be placed in 37 DEG C, 5%CO2Incubator in be incubated for 1h.
Ghost control group (CELL): serum free medium adds in hole after 37 DEG C of incubation 1.5h, continues to be placed in 37 DEG C,
1h is incubated in the incubator of 5%CO2.
3, after completing step 2,96 orifice plates are taken, abandon supernatant, DMEM+2% (volumn concentration) FBS culture medium (100 is added
The hole μ l/), 37 DEG C, 5%CO2 continues culture 1 week, until obvious lesion occurs in cell；Culture plate is taken out, under the microscope, is counted not
The cell hole count of lesion, parts of lesions and complete lesion, inhibiting rate (table 1) of the calculating antibody to adenovirus infection.
The anti-HAdV7 infection cell testing result of 1 3-3E of table
* concentration unit: μ g/mL；0h: inoculating cell after antibody and the total incubation of virus；+ 1h: after virus infected cell 1 hour
Add antibody
The result shows that the present invention, which prepares antibody 3-3E, can effectively inhibit the proliferation of HAdV7, EC50=0.4 when prevention uses
μ g/mL, EC50=0.72 μ g/mL when being used after virus infection.
Two, the anti-human 7 type adenovirus infection CB-17SCID mouse of 3-3E
1, Animal Model
It takes CB-17SCID mouse (dimension tonneau China), female, 16-18g, experimental day weighing, is randomly divided into experimental group and right
According to group (every group 12), isoflurane inhalation anesthesia, experimental group collunarium inoculation 3 step 1 of embodiment 2 in the HAdV7 virus for preparing
Concentrate (108TCID50/ is only), control group collunarium is inoculated with isometric culture medium.Timing daily is surveyed weight and is observed tested dynamic
Object depilation, feed, drinking-water and activity condition.
As a result as shown in Figure 6.Start within experimental mice virus inoculation second day to feed and drink water as the result is shown to reduce, hair becomes
Coarse, lean body mass, symptoms last 5 days or so, control group body did not had significant change, and continued weight increases, it was demonstrated that experimental group
Mouse infects ADV7 virus really.
2. the anti-influence to mouse is uninfected by of antibody 3-3E
It takes CB-17SCID mouse (dimension tonneau China), female, 16-18g, experimental day weighing, is randomly divided into experimental group and right
According to group (every group 12), isoflurane inhalation anesthesia dilutes 3-3E antibody prepared by embodiment 2 with plasma-free DMEM medium,
The antibody-solutions containing 200mg/ml 3-3E antibody, every 20 μ l of experimental group collunarium are obtained, control group is inoculated with isometric serum-free
Culture medium.Timing daily surveys weight and observes animal subject depilation, feed, drinking-water and activity condition.
As a result as shown in Figure 7.Body does not have significant change to experimental group compared with the control group as the result is shown, and continued weight increases
It is long, it was demonstrated that the inoculation of experimental group antibody will not generate mouse and significantly affect.
2, the anti-human 7 type adenovirus infection CB-17SCID mouse of 3-3E
It takes CB-17SCID mouse (dimension tonneau China), female, 16-18g, experimental day weighing, is randomly divided into experimental group and right
It is proceeded as follows according to group (every group 12):
Experimental group (3-3E): the 3-3E antibody of preparation is diluted with plasma-free DMEM medium, is obtained containing 200ug/ml
The antibody-solutions of 3-3E antibody；By the HAdV7 viral concentration liquid prepared in antibody-solutions and the 2 of 3 step 1 of embodiment, (virus is dense
Degree is 1010TCID50/ml it) is mixed according to volume ratio 1:1, mixes 37 DEG C of incubation 1h, experimental group prepared by collunarium inoculation step 1 is small
Mouse, every 20 μ l, the depilation of observation mouse, feed, drinking-water and activity condition simultaneously record mouse weight.
Control group (VIRUS-only): the HAdV7 viral concentration liquid (virus concentration that will be prepared in the 2 of 3 step 1 of embodiment
It is 1010TCID50/ml it) is mixed with plasma-free DMEM medium according to volume ratio 1:1,37 DEG C of incubation 1h, collunarium inoculation step 1 is made
Standby experimental mice (every inoculum concentration is 20 μ l), the depilation of observation mouse, feed, drinking-water and activity condition simultaneously record mouse
Weight.
As a result as shown in Figure 8.The results show that go out within control group second day the symptoms such as the new apparent bodily form is thin, and hair color is coarse,
And experimental mice symptom and control group comparison are substantially reduced, illustrate that 3-3E antibody can effectively mitigate mouse due to virus infection
The symptoms such as caused weight loss and hair color are coarse.
Sequence table
<110>PLA Academy of Military Sciences's military medical research institute
<120>7 type adenovirus monoclonal antibody 3-3E of people and its application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 354
<212> DNA
<213>artificial sequence (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 (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 (Artificial Sequence)
<400> 3
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
<211> 108
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
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 (10)
1. a kind of monoclonal antibody, including heavy chain variable region and light chain variable region；The heavy chain variable region includes that three complementations are determined
Determine area HCDR1, HCDR2 and HCDR3；The light chain variable region includes three complementary determining regions LCDR1, LCDR2 and LCDR3；
The amino acid sequence of the HCDR1 is following (a1) or (a2) or (a3):
(a1) sequence 2 of sequence table amino acid sequence shown in N-terminal 26-33；
(a2) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 26-33 passes through one or several amino acid residues
Substitution and/or the obtained amino acid sequence with the same function of deletion and/or addition；
(a3) with the amino acid sequence shown in N-terminal 26-33 of the sequence of sequence table 2 with 75% or 75% or more it is homologous
Property and amino acid sequence with the same function；
The HCDR2 is following (a4) or (a5) or (a6):
(a4) sequence 2 of sequence table amino acid sequence shown in N-terminal 51-58；
(a5) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 51-58 passes through one or several amino acid residues
Substitution and/or the obtained amino acid sequence with the same function of deletion and/or addition；
(a6) with the amino acid sequence shown in N-terminal 51-58 of the sequence of sequence table 2 with 75% or 75% or more it is homologous
Property and amino acid sequence with the same function；
The HCDR3 is following (a7) or (a8) or (a9):
(a7) sequence 2 of sequence table amino acid sequence shown in N-terminal 97-110；
(a8) by the sequence of sequence table 2, the amino acid sequence shown in N-terminal 97-110 is residual by one or several amino acid
The amino acid sequence with the same function that the substitution and/or deletion and/or addition of base obtain；
(a9) with the amino acid sequence shown in N-terminal 97-110 of the sequence of sequence table 2 with 75% or 75% or more it is same
Source property and amino acid sequence with the same function；
The amino acid sequence of the LCDR1 is following (b1) or (b2) or (b3):
(b1) sequence 4 of sequence table amino acid sequence shown in N-terminal 27-32；
(b2) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 27-32 passes through one or several amino acid residues
Substitution and/or the obtained amino acid sequence with the same function of deletion and/or addition；
(b3) with the amino acid sequence shown in N-terminal 27-32 of the sequence of sequence table 4 with 75% or 75% or more it is homologous
Property and amino acid sequence with the same function；
The amino acid sequence of the LCDR2 is following (b4) or (b5) or (b6):
(b4) sequence 4 of sequence table amino acid sequence shown in N-terminal 50-52；
(b5) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 50-52 passes through one or several amino acid residues
Substitution and/or the obtained amino acid sequence with the same function of deletion and/or addition；
(b6) with the amino acid sequence shown in N-terminal 50-52 of the sequence of sequence table 4 with 75% or 75% or more it is homologous
Property and amino acid sequence with the same function；
The amino acid sequence of the LCDR3 is following (b7) or (b8) or (b9):
(b7) sequence 4 of sequence table amino acid sequence shown in N-terminal 89-96；
(b8) by the sequence of sequence table 4, the amino acid sequence shown in N-terminal 89-96 passes through one or several amino acid residues
Substitution and/or the obtained amino acid sequence with the same function of deletion and/or addition；
(b9) with the amino acid sequence shown in N-terminal 89-96 of the sequence of sequence table 4 with 75% or 75% or more it is homologous
Property and amino acid sequence with the same function.
2. monoclonal antibody as described in claim 1, it is characterised in that:
The amino acid sequence of the heavy chain variable region is following (c1) or (c2) or (c3):
(c1) amino acid sequence shown in the sequence 2 of sequence table；
(c2) amino acid sequence shown in the sequence of sequence table 2 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or add obtained amino acid sequence with the same function；
(c3) homology with amino acid sequence shown in the sequence of sequence table 2 with 75% or 75% or more and have identical function
The amino acid sequence of energy；
The amino acid sequence of the light chain variable region is following (c4) or (c5) or (c6):
(c4) amino acid sequence shown in the sequence 4 of sequence table；
(c5) amino acid sequence shown in the sequence of sequence table 4 by the substitution of one or several amino acid residues and/or is lacked
Lose and/or add obtained amino acid sequence with the same function；
(c6) homology with amino acid sequence shown in the sequence of sequence table 4 with 75% or 75% or more and have identical function
The amino acid sequence of energy.
3. encoding the gene of antibody as claimed in claim 1 or 2.
4. gene as claimed in claim 3, it is characterised in that:
The gene of the heavy chain variable region of encoding said antibody is following (d1) or (d2) or (d3):
(d1) DNA molecular shown in the sequence 1 of sequence table；
(d2) nucleotide sequence limited with (d1) has 75% or 75% or more homology, and encodes institute in claims 1 or 2
The DNA molecular for the heavy chain variable region stated；
(d3) nucleotide sequence hybridization limited under strict conditions with (d1) or (d2), and encode described in claims 1 or 2
Heavy chain variable region DNA molecular；
The gene of the light chain variable region of encoding said antibody is following (d4) or (d5) or (d6):
(d4) DNA molecular shown in the sequence 3 of sequence table；
(d5) nucleotide sequence limited with (d4) has 75% or 75% or more homology, and encodes institute in claims 1 or 2
The DNA molecular for the heavy chain variable region stated；
(d6) nucleotide sequence hybridization limited under strict conditions with (d4) or (d5), and encode described in claims 1 or 2
Heavy chain variable region DNA molecular.
5. monoclonal antibody of any of claims 1 or 2 is in preparation for preventing and/or treating disease caused by adenovirus infection
Drug in application.
6. monoclonal antibody of any of claims 1 or 2 is preparing the application in product；The purposes of the product is following (e1)
And/or (e2):
(e1) inhibit adenovirus；
(e2) adenovirus is neutralized.
7. a kind of for preventing and/or treating the drug of disease caused by adenovirus infection, active constituent be claim 1 or
Monoclonal antibody described in 2.
8. a kind of product, active constituent is monoclonal antibody of any of claims 1 or 2；The purposes of the product is as follows
(e1) and/or (e2):
(e1) inhibit adenovirus；
(e2) adenovirus is neutralized.
9. such as application described in claim 5 or 6, or, drug as claimed in claim 7, or, product according to any one of claims 8,
It is characterized by: the adenovirus is adenovirus hominis.
10. application as claimed in claim 9 or drug or product, it is characterised in that: 7 type adenopathies of the adenovirus hominis behaviour
Poison.
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| CN110294802A (en) * | 2019-07-08 | 2019-10-01 | 中国人民解放军军事科学院军事医学研究院 | A kind of monoclonal antibody 10G12 and its application |
| WO2020192080A1 (en) * | 2019-03-28 | 2020-10-01 | 中国人民解放军军事科学院军事医学研究院 | Human adenovirus type 7 antibody and application thereof |
| CN116789807A (en) * | 2023-06-14 | 2023-09-22 | 珠海重链生物科技有限公司 | Anti-adenovirus monoclonal antibody and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942011A (en) * | 2010-01-19 | 2011-01-12 | 呼吸疾病国家重点实验室 | Neutralizing epitope of human adenovirus type 3 (HAdV-3) and type 7 (HAdV-7) and application thereof |
| CN104086650B (en) * | 2014-06-16 | 2017-01-04 | 广州锐达生物科技有限公司 | Neutralizing monoclonal antibody of people 7 type adenovirus and its preparation method and application |
| CN109957009B (en) * | 2019-03-28 | 2021-03-19 | 中国人民解放军军事科学院军事医学研究院 | Anti-human 7-type adenovirus antibody 2-1H and application thereof |
| CN109897104B (en) * | 2019-03-28 | 2020-12-22 | 中国人民解放军军事科学院军事医学研究院 | Human adenovirus 7 monoclonal antibody 3-3E and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020192080A1 (en) * | 2019-03-28 | 2020-10-01 | 中国人民解放军军事科学院军事医学研究院 | Human adenovirus type 7 antibody and application thereof |
| CN110294802A (en) * | 2019-07-08 | 2019-10-01 | 中国人民解放军军事科学院军事医学研究院 | A kind of monoclonal antibody 10G12 and its application |
| WO2021003867A1 (en) * | 2019-07-08 | 2021-01-14 | 中国人民解放军军事科学院军事医学研究院 | Monoclonal antibody 10g12 and application thereof |
| CN116789807A (en) * | 2023-06-14 | 2023-09-22 | 珠海重链生物科技有限公司 | Anti-adenovirus monoclonal antibody and application thereof |
| CN116789807B (en) * | 2023-06-14 | 2024-01-16 | 珠海重链生物科技有限公司 | Anti-adenovirus monoclonal antibody and application thereof |
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