CN109097337B - Hybridoma cell capable of secreting anti-Ad3FK monoclonal antibody, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a hybridoma cell capable of secreting an anti-Ad3FK monoclonal antibody, a monoclonal antibody, and a preparation method and application thereof. A hybridoma cell capable of secreting an anti-Ad3FK monoclonal antibody, wherein the preservation number is CCTCC No: C201875. the monoclonal antibody resisting Ad3FK secreted by the hybridoma cell has the advantages of high specificity, stable performance and the like, can be specifically combined with the human type 3 adenovirus, can be widely applied to preparation of medicaments for treating related diseases infected with the human type 3 adenovirus, and can also be used for preparation of detection reagents or detection equipment for detecting the human type 3 adenovirus.

Description

Hybridoma cell capable of secreting anti-Ad3FK monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a hybridoma cell capable of secreting an anti-Ad3FK monoclonal antibody, the monoclonal antibody, and a preparation method and application thereof.

Background

Human adenoviruses (HAdV) have been discovered to date with 7 groups of over 50 serotypes, more than 69 genotypes. Adenovirus is one of the main pathogens of pathogenic virulent pneumonia, and adenovirus of B group, C group and E group can cause respiratory tract infection, wherein adenovirus of 3 type, 7 type, 14 type, 55 type and E group 4 type of human adenovirus of B group are important pathogens causing acute respiratory tract infection (ARI) and infantile lethal pneumonia, and the infection is strong, and outbreak epidemic is often caused.

The epidemic strain of human adenovirus in China is mainly type 3 adenovirus, and particularly in infant adenovirus infection, the human type 3 adenovirus is the most main pathogen causing acute respiratory infection and severe pneumonia, and has strong infectivity, which often causes outbreak of epidemic. In addition, human type 3 adenoviruses also often cause ocular keratoconjunctivitis and pharyngoconjunctival fever.

Thus, the pathogenic hazard of human type 3 adenovirus makes its control important. However, no specific effective therapeutic drug exists for human type 3 adenovirus.

Disclosure of Invention

Based on the above, a hybridoma cell capable of secreting the anti-Ad3FK monoclonal antibody, a monoclonal antibody, and a preparation method and application thereof are needed.

A hybridoma cell capable of secreting an anti-Ad3FK monoclonal antibody, wherein the preservation number is CCTCC No: C201875.

in one embodiment, the anti-Ad3FK monoclonal antibody secreted by the hybridoma cells is labeled 1B 5.

An anti-Ad3FK monoclonal antibody, wherein the light chain of the anti-Ad3FK monoclonal antibody comprises an amino acid sequence shown as SEQ ID No.1, and the heavy chain of the anti-Ad3FK monoclonal antibody comprises an amino acid sequence shown as SEQ ID No. 2.

In one embodiment, the gene encoding the light chain of the anti-Ad3FK monoclonal antibody comprises the base sequence shown as SEQ ID No.3, and the gene encoding the heavy chain of the anti-Ad3FK monoclonal antibody comprises the base sequence shown as SEQ ID No. 4.

A medicament for treating adenovirus 3-associated diseases in humans, wherein said hybridoma is selected from the group consisting of the anti-Ad3FK monoclonal antibodies.

A detection reagent comprises the hybridoma cell or the anti-Ad3FK monoclonal antibody.

A preparation method of hybridoma cells capable of secreting anti-Ad3FK monoclonal antibody comprises the following steps:

mixing Ad3FK antigen with Freund's complete adjuvant at a volume ratio of 1:1 to obtain an emulsifier;

immunizing BALB/c mice with the emulsifier at the antigen dose of 40-80 mug/mouse, and feeding for 2-3 weeks to obtain primary immunized mice;

mixing the Ad11FK antigen and Freund's incomplete adjuvant according to a volume ratio of 1:1, performing boosting immunization on the primarily immunized mice at an antigen dose of 40-80 mug/mouse, and feeding for 2-3 weeks to obtain the boosted mice;

performing intrasplenic impact immunization on the mice subjected to the boosting immunization by using the Ad3FK antigen at the antigen dose of 20 to 50 mu g/mouse, and feeding for 2 to 4 days to obtain the mice subjected to the impact immunization;

collecting splenocytes from the shock immunized mouse; and

fusing the splenocytes with mouse myeloma cells to obtain fused cells; and

screening the fusion cells by using the Ad3FK antigen to obtain positive fusion cells, and cloning the positive fusion cells to obtain hybridoma cells stably secreting 1B 5.

In one embodiment, the step of fusing the spleen cells with mouse myeloma cells to obtain fused cells comprises:

respectively sucking the spleen cells and the mouse myeloma cells into a centrifuge tube, adding an incomplete culture solution, uniformly mixing, centrifuging, and removing supernatant to obtain a precipitation solution, wherein the number ratio of the spleen cells to the mouse myeloma cells is 5-3: 1; and

adding a PEG fusion agent solution into the precipitation solution, standing for 80-100 s, and adding an incomplete culture solution to terminate fusion to obtain the fused cells, wherein the ratio of the PEG fusion agent solution to the splenocytes is 1 mL: 10⁸And (4) respectively.

In one embodiment, the step of screening the fusion cells with the Ad3FK antigen to obtain positive fusion cells and cloning the positive fusion cells to obtain hybridoma cells stably secreting 1B5 comprises:

culturing the fusion cell, and screening by using an indirect enzyme-linked immunosorbent assay to obtain the positive fusion cell; and

and (3) adopting a limiting dilution method to perform monoclonality on the positive fusion cells to obtain hybridoma cells stably secreting 1B 5.

Experimental results show that the monoclonal antibody resisting Ad3FK secreted by the hybridoma has the advantages of high specificity, stable performance and the like, can be specifically combined with the human type 3 adenovirus, can be widely applied to preparation of medicaments for treating related diseases infected with the human type 3 adenovirus, and can also be used for preparation of detection reagents or detection equipment for detecting the human type 3 adenovirus. Can realize accurate and rapid detection of human type 3 adenovirus in blood, and is beneficial to early diagnosis of human type 3 adenovirus related diseases.

The hybridoma cell or the anti-Ad3FK monoclonal antibody is applied to preparation of medicaments for treating diseases related to human type 3 adenovirus infection.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of Ad11FK antigen of example 1;

FIG. 2 is a graph of the results of cross-reactivity of antisera to the Ad3, Ad4, Ad7, Ad11, Ad14, Ad55 virus of the Ad11FK antigen of example 1;

FIG. 3 is a graph showing the results of detection of monoclonal antibodies 1A12, 3A9, 1B5, 2B8 and 7F10 by the indirect ELISA method of example 4;

FIG. 4 is a graph showing the results of neutralization of different types of adenovirus by the monoclonal antibody of example 4;

FIG. 5 is a graph showing the results of the inhibition of Ad3EGFP virus by monoclonal antibody 1B5 of example 4;

FIG. 6 is a graph showing the results of cross-reaction of monoclonal antibody 1B5 of example 4 with recombinant knob protein of a different type of adenovirus;

FIG. 7 is a cross-reaction of monoclonal antibody 1B5 of example 4 with adenovirus particles of different types;

FIG. 8 is a graph showing the results of Western blot detection of the reaction of monoclonal antibody 1B5 of example 4 with different types of adenoviruses;

FIG. 9 is a sequence analysis diagram of the variable region in the light chain of monoclonal antibody 1B5 of example 4;

FIG. 10 is a diagram showing the sequence analysis of the heavy chain variable region of monoclonal antibody 1B5 of example 4.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the hybridoma cells secreting the anti-Ad3FK monoclonal antibody is deposited at the chinese type culture collection center (CCTCC) at 29/6/2018, address: china, Wuhan university, the preservation number is CCTCC No: c201875, name by classification: hybridoma cell line 1B 5. The hybridoma cells secreted monoclonal antibodies against Ad3FK as 1B 5. 1B5 can be used as a detection antibody, and 1B5 can be applied to the preparation field of a detection reagent for human adenovirus type 3 or a detection device for human adenovirus type 3. Can also be applied to the preparation of medicaments for treating the diseases related to the infection of the human type 3 adenovirus.

The method for preparing a hybridoma capable of secreting an anti-Ad3FK monoclonal antibody according to one embodiment includes the following steps S110 to S170.

S110, preparing Ad3FK antigen.

Specifically, the procedure for preparing Ad3FK antigen includes S111 to S117.

S111, carrying out codon optimization on the gene of the head structural domain (fiber knob) of the cilia protein of the human type 3 adenovirus so as to adapt to the expression of escherichia coli.

Furthermore, the sequence of the gene of the head structural domain (fiber knob) of the optimized human type 3 adenovirus is shown in SEQ ID No. 5, and the amino acid sequence of the head structural domain of the optimized human type 3 adenovirus is shown in SEQ ID No. 6.

S113, artificially synthesizing the optimized sequence of the gene of the head structural domain (fiber knob) of the cilia protein of the human adenovirus type 3, amplifying and cloning the sequence into an expression vector to obtain a recombinant expression vector.

Further, after amplifying the sequence of the gene of the cilia protein (fiber knob) of the artificially synthesized and optimized human adenovirus type 3, an amplification product is obtained. And (3) carrying out double digestion on the amplified product by using BamHI and HindIII, and then connecting the amplified product to a prokaryotic expression vector which is subjected to double digestion by using BamHI and HindIII to obtain a recombinant expression vector.

Further, the prokaryotic expression vector is PQE 30.

And S115, transforming the recombinant expression vector into escherichia coli, and screening and identifying to obtain the recombinant engineering bacteria.

Further, Escherichia coli is Escherichia coli BL21(DE 3).

And S117, carrying out induction culture on the recombinant engineering bacteria to obtain the Ad3FK antigen.

Specifically, 0.1 mM-1 mM IPTG is adopted, recombinant engineering bacteria are induced at the low temperature of 18-30 ℃, cultured overnight, and bacteria are collected. Then, the recombinant engineering bacteria are resuspended in PBS buffer (1mM EDTA, 10% glycerol, 1% Triton X100, 10mM imidazole), lysed by lysozyme, and the supernatant is taken after high-speed centrifugation to obtain the initial product. The initial product was then purified to give purified Ad3FK antigen.

Further, purification of the crude product includes purification with a nickel column, washing and elution with gradient imidazole, and centrifugation in a 10kDa ultrafiltration tube to remove imidazole, resulting in purified Ad3FK antigen.

The Ad3FK antigen is not limited to the one produced by the method of the present embodiment, and may be produced by a production method that is conventional in the art, and a commercial Ad3FK antigen may be purchased.

And S120, carrying out primary immunization on the mice to obtain the mice subjected to primary immunization.

Specifically, after Ad3FK antigen was emulsified, BALB/c mice were immunized with 40. mu.g/mouse to 80. mu.g/mouse of antigen, and the mice were raised for 2 weeks to 3 weeks to obtain primary-immunized mice. The antigen dose refers to the content of Ad3FK antigen.

Specifically, Ad3FK antigen was mixed with freund's complete adjuvant at a volume ratio of 1:1 to give an emulsifier. BALB/c mice were immunized with 40. mu.g/mouse to 80. mu.g/mouse of antigen, and the mice were raised for 2 to 3 weeks to obtain primary-immunized mice.

S130, performing booster immunization on the primarily immunized mice to obtain boosted mice.

Specifically, Ad3FK antigen was emulsified, and the primary immunized mice were boosted at an antigen dose of 40 to 80. mu.g/mouse, and then raised for 2 to 3 weeks to obtain boosted mice.

Specifically, the Ad11FK antigen was mixed with Freund's incomplete adjuvant, and the primary immunized mice were boosted at an antigen dose of 40 to 80 μ g/mouse, and then raised for 2 to 3 weeks to obtain boosted mice.

Of course, in other embodiments, Ad3FK antigen may also be mixed with incomplete freund's adjuvant according to a ratio of 1:1, and then the mixture is boosted at a dose of 40 to 80. mu.g/mouse, and the boosted mice are obtained after 2 to 3 weeks of rearing.

Further, the booster immunization is carried out for a plurality of times, and the time interval between adjacent booster immunizations is 2 weeks to 3 weeks

Further, the booster immunization is carried out twice, and the time interval of the two booster immunizations is 2 weeks to 3 weeks.

And S140, performing impact immunization on the mice subjected to the enhanced immunization to obtain the mice subjected to the impact immunization.

Specifically, the mice which are boosted with Ad3FK antigen are subjected to intrasplenic shock immunization at an antigen dose of 20 to 50. mu.g/mouse, and the mice which are shock-immunized are obtained after 2 to 4 days of feeding.

S150, collecting splenocytes of the impact-immunized mice.

And S160, fusing the spleen cells and the mouse myeloma cells to obtain fused cells.

Specifically, the procedure of fusing spleen cells with mouse myeloma cells to obtain fused cells includes S161 to S163.

S161, respectively sucking splenocytes and mouse myeloma cells into a centrifuge tube, adding the incomplete culture solution, uniformly mixing, centrifuging, and removing supernatant to obtain a precipitation solution. Wherein the number ratio of the splenocytes to the mouse myeloma cells is 5-3: 1.

preferably, the ratio of the number of splenocytes to mouse myeloma cells is 5: 1.

further, the mouse myeloma cell is a mouse myeloma cell SP 2/0.

Further, the incomplete culture solution is a serum-free culture solution. In this embodiment, the incomplete culture medium is a serum-free RPMI-1640 culture medium. In other embodiments, the incomplete culture solution may be a serum-free DMEM culture solution or an IMDM culture solution.

Furthermore, the centrifugal speed is 1000 rpm-1500 rpm, and the centrifugal time is 5 min-10 min.

And S163, adding a PEG fusion agent solution into the precipitation solution, standing for 80-100S, and adding an incomplete culture solution to terminate fusion to obtain the fused cells. Wherein, the proportion of the PEG fusion agent solution to the splenocytes is 1 mL: 10⁸And (4) respectively.

Further, the PEG fusion agent is PEG4000 fusion agent.

S170, screening the fusion cells by using Ad3FK antigen to obtain positive fusion cells, and cloning the positive fusion cells to obtain hybridoma cells stably secreting 1B 5.

Specifically, the procedures of screening fusion cells by using Ad3FK antigen to obtain positive fusion cells, cloning the positive fusion cells to obtain hybridoma cells stably secreting 1B5 include S171 to S173.

S171, screening the fusion cells by using Ad3FK antigen to obtain positive fusion cells.

Specifically, the fused cells are cultured to an appropriate concentration, and positive fused cells are obtained by screening with an indirect enzyme-linked immunosorbent assay.

Further, coating the Ad3FK antigen on an enzyme label plate, sealing with a sealing solution, adding a fusion cell culture supernatant, incubating for 0.5 h-1 h, washing, patting to dry, adding a second antibody, incubating for 0.5 h-1 h, washing again, patting to dry, adding a color developing agent, developing, measuring an absorbance value by an enzyme label analyzer, and screening to obtain positive fusion cells.

Further, the secondary antibody was horseradish peroxidase-labeled goat anti-mouse IgG.

S173, cloning the positive fusion cells to obtain hybridoma cells stably secreting 1B 5.

Specifically, after culturing the fused cells to an appropriate cell concentration, the positive fused cells were monoclonalized by the limiting dilution method to obtain hybridoma cells stably secreting 1B 5.

In the embodiment, a hybridoma cell capable of stably secreting 1B5 is obtained by screening, and the preservation number is CCTCC No: C201875.

of course, in practical applications, the preparation of hybridoma cells is not limited to the sequence of steps S110 to S170, and can be adjusted as required by those skilled in the art.

In the preparation of monoclonal antibodies, the conventional method is to use whole adenovirus particles as immunogen and whole adenovirus particles as detection antigen to prepare neutralizing monoclonal antibodies, but the whole adenovirus particles as immunogen and detection antigen are usually used to obtain monoclonal antibodies aiming at hexon protein, and neutralizing monoclonal antibodies of cilium protein as adenovirus receptor binding protein cannot be obtained. It is proved that the hybridoma cell prepared by the method can continuously and efficiently secrete the neutralizing monoclonal antibody of the cilium protein, and the method for preparing the hybridoma cell is simple and easy to implement.

One embodiment of an anti-Ad3FK monoclonal antibody, the light chain comprises the amino acid sequence set forth in SEQ ID No.1 and the heavy chain comprises the amino acid sequence set forth in SEQ ID No. 2.

In one embodiment, the gene encoding the light chain of the anti-Ad3FK monoclonal antibody comprises the base sequence shown in SEQ ID No.3, and the gene encoding the heavy chain of the anti-Ad3FK monoclonal antibody comprises the base sequence shown in SEQ ID No. 4.

The anti-Ad3FK monoclonal antibody can be specifically combined with human type 3 adenovirus, and can be applied to preparation of medicaments for treating diseases related to human type 3 adenovirus infection. The kit can also be used for preparing a detection reagent or detection equipment for detecting the human adenovirus type 3, can realize accurate and rapid detection of the human adenovirus type 3 in blood, and is favorable for early diagnosis of human adenovirus type 3 related diseases.

A medicament for treating diseases related to human type 3 adenovirus infection comprises the hybridoma cells or the anti-Ad3FK monoclonal antibody.

In one embodiment, the above medicament for treating human type 3 adenovirus-associated diseases further comprises a pharmaceutically acceptable adjuvant or carrier.

Human adenovirus is a non-enveloped icosahedral symmetric structure, and the main capsid proteins comprise hexon and penton, and the penton comprises penton base (penton base) and cilium protein (fiber), which can induce specific neutralizing antibody response in vivo. The hexon is traditionally considered to be the most important neutralizing antigen, while the cilin is the minor neutralizing antigen. The medicine for treating the diseases related to the human type 3 adenovirus comprises the monoclonal antibody resisting Ad3FK secreted by the hybridoma cell or the monoclonal antibody resisting Ad3FK, and the monoclonal antibody resisting Ad3FK can be combined with the human type 3 adenovirus, so that the head structural domain of cilia protein of the human type 3 adenovirus is prevented from being combined with a specific receptor of the human type 3 adenovirus on the surface of a host cell, the human type 3 adenovirus cannot be adsorbed, the human type 3 adenovirus is neutralized, and the effect of eliminating the human type 3 adenovirus is achieved.

A detection reagent comprising the hybridoma cell or the anti-Ad3FK monoclonal antibody.

In one embodiment, the detection reagent further comprises an auxiliary detection reagent.

It can be understood by those skilled in the art that the anti-Ad3FK monoclonal antibody of the present embodiment can be directly or indirectly combined with other signal groups (such as magnetic microspheres, horseradish peroxidase, etc.), or the anti-Ad3FK monoclonal antibody of the present embodiment can be used as a coating antibody (such as ELISA), so that the anti-Ad3FK monoclonal antibody can be widely used in the field of detection of human adenovirus type 3.

The following are specific examples.

The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits.

The GenBank accession number of the Ad3GZ01 virus strain in the examples of the present specification is DQ099432, hereinafter abbreviated as Ad 3. The GenBank accession number of the Ad4GZ01 virus strain is KF006344.1, hereinafter abbreviated Ad 4. GenBank accession number of the Ad11Slobitski strain is AF532578.1, hereinafter abbreviated as Ad 11. The GenBank accession number of the Ad7GZ08 virus strain is GQ478341.1, abbreviated as Ad7 hereinafter. The GenBank accession number of the Ad14p1GZ01 virus strain is JQ824845.1, abbreviated as Ad14 hereinafter. The GenBank accession number of the Ad55Shanxi-Y16 virus strain is KF911353.1, hereinafter abbreviated as Ad 55. AD293 cells were purchased from the biological resource center (ATCC).

Example 1

Preparation of Ad3FK antigen

(1) The gene of the head structural domain (fiber knob) of the cilia protein of the human type 3 adenovirus is subjected to codon optimization, the sequence SEQ ID No. 5 of the optimized gene of the head structural domain (fiber knob) of the cilia protein of the human type 3 adenovirus is shown, and the amino acid sequence of the head structural domain of the optimized cilia protein of the human type 3 adenovirus is shown in SEQ ID No. 6.

(2) After amplifying the sequence of the gene of the head domain (fiber knob) of the ciliated protein of the artificially synthesized and optimized human adenovirus type 3, an amplification product is obtained. The amplified product was double-digested with BamHI and HindIII and ligated to a prokaryotic expression vector PQE30 (Qiagen) which had been double-digested with BamHI and HindIII to obtain a recombinant expression vector.

(3) The recombinant expression vector was transformed into E.coli BL21(DE3) and smeared in a medium containing Kan^rCulturing on a flat plate for 12h, observing whether the monoclonal grows, and if the monoclonal grows, primarily judging that the transformation is successful. And then selecting the transformant to carry out colony PCR further verification, and screening to obtain the successfully transformed recombinant engineering bacteria.

(4) Inducing the recombinant engineering bacteria at 18 ℃ by adopting 0.5mM IPTG, culturing overnight, and collecting bacteria. Then, the recombinant engineering bacteria are resuspended in PBS buffer (1mM EDTA, 10% glycerol, 1% Triton X100, 10mM imidazole), lysed by lysozyme, and the supernatant is taken after high-speed centrifugation to obtain the initial product.

(5) Purification with a nickel column, gradient imidazole washing and elution, centrifugation in a 10kDa ultrafiltration tube to remove imidazole and obtain purified Ad3FK antigen, SDS-PAGE analysis of the purified Ad3FK antigen, in particular: two portions of purified Ad3FK were added to the electrophoresis buffer (containing 2% SDS, 1% mercaptoethanol), and after 5 minutes at room temperature, they were placed on ice and heated at 98 ℃ for 5 minutes for loading, electrophoretically separated in 12% SDS-PAG, and stained with Coomassie brilliant blue, and the results are shown in FIG. 1. In FIG. 1, lane M (numbered M in the figure) is protein molecular weight marker, the first lane (numbered 1 in the figure) is Ad3FK antigen treated on ice for 5min, and the second lane (numbered 2 in the figure) is Ad3FK antigen treated with heat at 98 ℃ for 5 min.

(6) Adding aluminum phosphate adjuvant (1: 1) into Ad3FK antigen, emulsifying, immunizing BALB/c mouse for four times, immunizing each mouse with 40ug protein each time, collecting serum 7 days after last immunization, and cross reacting Ad3FK antigen (1: 2000) with Ad3, Ad4, Ad7, Ad11, Ad14 and Ad55 viruses; sera from mice immunized with PBS (1: 2000) served as negative controls, and sera from mice immunized with Ad3 virus served as positive controls. Performing ELISA detection. The results are shown in FIG. 2. In FIG. 2, anti-Ad3FK refers to the serogroup of mice immunized with Ad3FK antigen, anti-Ad3 refers to the positive control group, and anti-PBS refers to the negative control group.

As can be seen from fig. 1, Ad3FK antigen is mainly multimeric when unheated, and is expressed as monomeric when heated.

As shown in FIG. 2, the serum of mice immunized with Ad3FK antigen recognized Ad3, Ad7, Ad14 and Ad55, and showed weak response to Ad4 and Ad 11.

Example 2

Preparation of Ad4FK, Ad7FK, Ad11FK, Ad14FK and Ad55FK

The head domain of cilia protein of adenovirus type 4 (Ad4FK), the head domain of cilia protein of adenovirus type 7 (Ad7FK), the head domain of cilia protein of adenovirus type 11 (Ad11FK), the head domain of cilia protein of adenovirus type 14 (Ad14FK) and the head domain of cilia protein of adenovirus type 55 (Ad55FK) were prepared in substantially the same manner as the Ad3FK antigen in example 1, except that the genes encoding the respective proteins were different from the Ad3FK antigen and the amino acids constituting the respective proteins were different from the Ad3FK antigen. Specifically, the gene sequence of the code Ad4FK is shown as SEQ ID No. 7, and the amino acid sequence is shown as SEQ ID No. 8. The gene sequence of the code Ad7FK is shown as SEQ ID No. 9, and the amino acid sequence is shown as SEQ ID No. 10. The gene sequence of the coding Ad11FK is shown as SEQ ID No. 11, and the amino acid sequence is shown as SEQ ID No. 12. The gene sequence of the coding Ad14FK is shown as SEQ ID No. 13, and the amino acid sequence is shown as SEQ ID No. 14. The gene sequence of the coding Ad55FK is shown as SEQ ID No. 15, and the amino acid sequence is shown as SEQ ID No. 16.

Example 3

Preparation of hybridoma capable of secreting anti-Ad3FK monoclonal antibody

(1) BALB/c mice (BALB/c mice purchased from Guangdong provincial medicine laboratory animal center) were immunized at a dose of 50. mu.g per mouse for primary immunization after emulsification with an equal volume of Freund's complete adjuvant in the Ad3FK antigen obtained in example 1. Two weeks after feeding, mice were immunized initially.

(2) After emulsification with an equal volume of Freund's incomplete adjuvant in the Ad3FK antigen obtained in example 1, booster immunization was performed at a dose of 50. mu.g each. Two weeks later, booster immunization was performed once more at the same dose, and two weeks later, booster mice were obtained.

(3) The Ad3FK antigen obtained in example 1 was used to immunize mice with intrasplenic shock immunization at a dose of 50 μ g per mouse, and the mice were raised for three days to obtain shock-immunized mice.

(4) Collecting spleens of immunized mice, and separating to obtain splenocytes.

(5) Respectively sucking splenocytes and mouse myeloma cells SP2/0 into a centrifuge tube, adding incomplete culture solution to 30mL, mixing uniformly, centrifuging at 1000rpm for 10min, and discarding supernatant to obtain a precipitate. Wherein the number ratio of splenocytes to mouse myeloma cells is 1 × 10⁸The method comprises the following steps: 3X 10⁷And (4) respectively. 1mL of PEG4000 fusogenic solution (added within 60 s) was added to the pellet, and after standing for 90s, 4mL of incomplete medium was added to terminate the fusion (3min was added), and the pellet was centrifuged at 800rpm for 7min, and the supernatant was discarded, 100mL of HAT medium was added, and the pellet was gently resuspended. The cell suspension was added to a 96-well plate, 0.1ml per well (equivalent to 2 drops), which had been plated with a feeder cell layer. The plate was then incubated at 37 ℃ in an incubator containing 5% CO2 to obtain fused cells.

(6) The Ad3FK antigen obtained in example 1 was diluted with a coating solution (pH 9.6, 0.05M carbonate buffer) to about 2. mu.g/mL, 100. mu.L per well was added to a 96-well plate ELISA plate (brand: Nunc Maxisorp), and the plate was coated overnight at 4 ℃. The coated 96-well plates were washed once with PBS (PBST) containing 0.05% Tween-20, and 100. mu.L of blocking solution (PBS containing 3% BSA) was added to each well and blocked at 37 ℃ for 2 h. After washing with PBST 1 time, 100. mu.L of fused cell culture supernatant was added to each well and incubated at 37 ℃ for 50 min. Thereafter, the wells were washed 5 times with PBST, and 100. mu.L of antibody dilution (PBST containing 2% BSA) 1: a10000-diluted horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody (Bio-Rad, China) was incubated at 37 ℃ for 50 min. Then, the wells were washed with PBST for 5 times, and 100. mu.L of TMB developing solution was added to each well to develop the color for 5 min. Finally, 30. mu.L of 2M sulfuric acid was added to each well to terminate the reaction, and the absorbance at 450nm was read in a microplate reader (model: Thermo Scientific Multiskan MK 3). Sera from mice immunized with Ad3FK antigen and sera from non-immunized mice (negative sera) were diluted with antibody 1: 10000 dilution, the same procedure, as positive and negative controls for each experiment. And determining the positive cell wells when the OD value reaches 0.2 or the ratio of the OD value to the OD value of the negative control wells is more than 2.

(7) Monoclonal cloning was performed on antibody-secreting positive cell wells by limiting dilution, the reactivity of monoclonal cell culture supernatants to the Ad3FK antigen from example 1 was examined by indirect ELISA, positive wells were selected and cloned four times in succession by the above method, and expanded culture was performed to obtain monoclonal hybridoma cell lines. The cell strain is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 6 and 29 months, and the address is as follows: china, Wuhan university, the preservation number is CCTCC No: c201875, name by classification: hybridoma cell line 1B 5.

Example 4

Preparation of monoclonal antibodies

The hybridoma cells obtained in example 3 were injected intraperitoneally into mice primed with Freund's incomplete adjuvant, and ascites were induced and collected. The antibody subclass of the mAb in ascites was determined using a mouse antibody subclass kit (Roche, USA), and the specific procedures were performed according to the kit instructions. The titer of ascites fluid against the Ad3FK antigen obtained in example 1 was determined by indirect ELISA with the highest dilution of the absorbance value of the negative control being greater than or equal to 2.1 times as the end point of the titer.

The ascites titer of the Ad3FK antigen obtained in example 1 was 10 by indirect ELISA method⁶。

Through indirect ELISA screening, 5 monoclonal antibodies against Ad3FK, namely 1A12, 3A9, 1B5, 2B8 and 7F10, are obtained. The results of detecting ascites (1: 10000 and 1: 1000 dilution) of 5 monoclonal antibodies by indirect ELISA are shown in FIG. 3.

Example 5

Screening of anti-human Ad3 neutralizing monoclonal antibody and specificity analysis thereof

(1) In vitro micro-neutralization experiments detect the neutralization effect of the monoclonal antibody on different types of adenoviruses. The method specifically comprises the following steps: after the ascites fluid obtained in example 4 was diluted 2-fold with DMEM medium, 50. mu.l of each dilution was mixed with 50. mu.l of each dilution containing 100TCID50 of Ad3GZ01 virus strain, Ad7GZ08 virus strain, Ad14p1GZ01 virus strain, Ad55Shanxi-Y16 virus strain and Ad11Slobitski virus strain, and incubated at 37 ℃ for 1 hour. Then adding the antibody-virus mixed solution into a monolayer AD293 cell with 85% -95% abundance cultured by a 96-well culture plate. And after 48h, observing and judging the neutralization titer by an inverted microscope, wherein the highest dilution capable of protecting the monolayer cells from forming obvious cytopathic effect is taken as a neutralization titer endpoint. PBS immunized mouse sera were used as negative controls for each experiment. The results are shown in FIG. 4. In FIG. 4 Ad3V corresponds to the Ad3GZ01 virus group, Ad7V corresponds to the Ad7GZ08 virus group, Ad14V corresponds to the Ad14p1GZ01 virus group, Ad55V corresponds to the Ad55Shanxi-Y16 virus group, and Ad11V corresponds to the Ad11Slobitski virus group.

(2) The inhibition effect of the monoclonal antibody 1B5 on the Ad3EGFP virus is detected. The method specifically comprises the following steps: the monoclonal antibody 1B5 which is diluted in a gradient manner and a recombinant human adenovirus type 3 Ad3EGFP (Ad3EGFP is prepared by inserting an EGFP expression frame in a human adenovirus type 3 genome E3 region through a conventional molecular cloning method, and Ad3EGFP can be expressed to generate green fluorescent protein) with green fluorescent protein (EGFP) of 100TCID50 are mixed, incubated for 1h, inoculated to AD293 cells, and observed and photographed under a fluorescent microscope after 48 h. Among them, the wells inoculated with 100TCID50Ad3EGFP without mab served as virus controls, and the wells without virus served as cell controls. The results are shown in FIG. 5.

(3) The cross-reaction of monoclonal antibody 1B5 with recombinant knob protein of different adenovirus types was detected by indirect ELISA. The method specifically comprises the following steps: different types of adenovirus recombinant knob proteins (Ad 3FK obtained in example 1 and Ad4FK, Ad7FK, Ad11FK, Ad14FK and Ad55FK obtained in example 2) were used as coating antigens, and indirect ELISA was performed, and the results are shown in FIG. 6.

(4) The cross-reaction of monoclonal antibody 1B5 with adenovirus particles of different types was detected by indirect ELISA. Different types of whole virus particles (Ad3, Ad4, Ad7, Ad11, Ad14 and Ad55) are used as coating antigens to carry out indirect ELISA detection, and the detection result is shown in FIG. 7. In fig. 7, Ad3V corresponds to Ad3GZ01 virome, Ad4V corresponds to Ad4GZ01 virome, Ad7V corresponds to Ad7GZ08 virome, Ad11V corresponds to Ad11Slobitski virome, Ad14V corresponds to Ad14p1GZ01 virome, and Ad55V is Ad55Shanxi-Y16 virome.

(5) Immunoblotting experiments were performed with different types of adenovirus recombinant knob proteins and purified whole virus particles as antigens. The results are shown in FIG. 8. In FIG. 8, lanes 1-5 are the head domains of ciliated proteins, and lanes 6-9 are whole virus particles; 1 corresponds to group Ad7FK, 2 corresponds to group Ad55FK, 3 corresponds to group Ad11FK, 4 corresponds to group Ad3FK for denaturation, 5 corresponds to group Ad3FK, 6 corresponds to group Ad3, 7 corresponds to group Ad3 for inactivation, 8 corresponds to group Ad7, 9 corresponds to group Ad 55; m indicates the molecular weight of the quasi-prestained protein (NEB), and is 245kDa, 190kDa, 130kDa, 100kDa, 80kDa (red), 58kDa, 46kDa, 32kDa, 25kDa (green) and 17kDa in this order.

As shown in FIG. 4, monoclonal antibody 1B5 in ascites has strong neutralizing activity against Ad3, ascites neutralizing potency exceeding 1/5120, and no neutralizing effect against other types in group B, such as Ad7, Ad14, Ad11, Ad55, etc.

As can be seen from FIG. 5, the cell trace neutralization experiment of the recombinant human type 3 adenovirus Ad3EGFP with the EGFP fluorescent protein further proves the neutralization effect of the monoclonal antibody 1B5 on the type 3 adenovirus, the monoclonal antibody 1B5 still has an obvious inhibition effect on the Ad3EGFP virus when diluted by 81920 times, and the fluorescence inhibition rate is more than 50%.

As can be seen from FIG. 6, monoclonal antibody 1B5 specifically binds to the fibrin head domain of adenovirus type 3, and is a biospecific antibody for adenovirus type 3, which does not recognize or responds very weakly to the fibrin head domain of other adenoviruses.

As can be seen from FIG. 7, monoclonal antibody 1B5 is biospecific for adenovirus type 3 and is not recognized or poorly reactive with other types of adenovirus.

As can be seen from fig. 8, monoclonal antibody 1B5 is an antibody specific for adenovirus type 3, recognizes only the cilia protein or the head domain of the cilia protein of adenovirus type 3 in the form of a 3-mer, does not recognize the cilia protein or the head domain of the cilia protein of Ad3 in the form of a denatured monomer, and does not react with other types of adenoviruses such as Ad7, Ad11, Ad55, and the like.

Example 6

Sequencing analysis of antibody gene sequence of hybridoma cell strain 1B5

(1) Extracting mRNA of hybridoma cell strain 1B5, reverse transcribing cDNA with 5' RACE, PCR to obtain antibody heavy chain/light chain variable region gene, cloning and sequencing heavy chain and light chain variable region (V) and hinge region (J), analyzing and sequencing result with bioinformatics, eliminating non-functional antibody gene, and determining possible CDR sequence.

Bioinformatics analysis results show that the light chain of the monoclonal antibody 1B5 is kappa-type, the gene encoding the light chain has a base sequence shown as SEQ ID No.3, and the amino acid sequence of the light chain is shown as SEQ ID No. 1. The heavy chain of the monoclonal antibody 1B5 is IgG type, the gene coding the light chain is shown as the base sequence of SEQ ID NO.4, and the amino acid sequence of the light chain is shown as SEQ ID NO. 2.

(2) The gene sequence, amino acid sequence, heavy chain gene sequence and amino acid sequence of the light chain were compared in the antibody database, and the comparison results are shown in fig. 9 and fig. 10.

FIG. 9 shows a comparison of the results showing that the V region gene and allele of the light chain of monoclonal antibody 1B5 is most similar to Musmus IGKV6-32 x 01F, up to 97.49% (272/279nt), with four amino acid differences; the J region gene Musmus IGKJ 2-01F has the highest similarity, reaching 97.37% (37/38nt) and has no amino acid difference. The 4 FR regions (FR-IMGT) are respectively 26.17.36.10 amino acids, the 3 CDR regions (FR-IMGT) are respectively 6.3.9 amino acids, and the AA JUNCTION sequence is CQQDYSSPYTF.

The comparison in FIG. 10 shows that the monoclonal anti-1B 5 heavy chain V region gene and allele has the highest similarity to Musmus IGHV 9-3-1X 01F, up to 98.96% (285/288nt), with 3 amino acid differences; the J region gene has the highest similarity with Musmus IGHJ 2X 01F, and reaches 93.75% (45/48nt), and no amino acid difference exists; the D region is the first reading frame (Musmus IGHD 3-1X 01F). The 4 FR regions (FR-IMGT) have 25.17.38.11 amino acids respectively, the 3 CDR regions (FR-IMGT) have 8.8.12 amino acids respectively, and the AA JUNCTION sequence is CARTAGARGYFDYW.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Guangzhou medical university affiliated first hospital

<120> hybridoma cell capable of secreting anti-Ad3FK monoclonal antibody, preparation method and application thereof

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Met Lys Ser Gln Thr Gln Val Phe Val Phe Leu Leu Leu Cys Val Ser

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Met Ile Asn Asp Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro

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Thr Val Gln Ala Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr

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Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

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Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

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Ala Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

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Thr Asn Phe Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

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Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

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Tyr Phe Cys Ala Arg Thr Ala Gly Ala Arg Gly Tyr Phe Asp Tyr Trp

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Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro

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<213> Artificial Sequence (Artificial Sequence)

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aaatacatca ggcaggcaag ggcatcaaga tgaagtcaca gacccaggtc ttcgtatttc 60

tactgctctg tgtgtctggt gctcatggga ctattgtgat gacccagact cccaaattcc 120

tgcttgtatc agcaggagac aggattacca taacctgcaa ggccagtcag agtatgatta 180

atgatgtagc ttggtaccaa cagaagccag gtcagtctcc taaactgctg atatactatg 240

catccaatcg ctacactggg gtccctgatc gcttcactgg cagtggatat gggacggatt 300

tcactttcac catcagcact gtgcaggctg aagacctggc agtttatttc tgtcagcagg 360

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ctgcaccaac tgtatcc 437

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ataccagcaa gcgagtgacc agttagtctt aaggcaccac ttcttagaca tcatggcttg 60

ggtgtggacc ttgctattcc tgatggcagc tgcccaaagt gcccaagcac agatccagtt 120

ggtgcagtct ggacctgagc tgaagaagcc tggagagaca gtcaagatct cctgcaaggc 180

ttctgggtat accttcacaa actttggaat gaactgggtg aagcaggctc caggaaaggg 240

tttaaagtgg atggcctgga taaacaccta cactggagag ccaaaatatg ctgatgactt 300

caagggacgg tttgccttct ctttggaaac ctctgccagc actgcctatt tgcagatcaa 360

caacctcaaa aatgaggaca cggctacata tttctgtgca agaacggctg gggccagggg 420

ttactttgac tattggggcc aaggcaccac tctcacagtc tcctcagcca aaacgacacc 480

cccatctgtc tatccactgg cccctg 506

<210> 5

<211> 588

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatagcattg cactgaaaaa taacaccctg tggaccggtc cgaaaccgga agccaattgt 60

attattgaat atggcaaaga aaatccggat agtaaactga ccttaattct ggttaaaaat 120

ggcggcattg ttaatggcta tgtgaccctg atgggcgcct cagattatgt taataccctg 180

tttaaaaaca aaaatgtgag cattaatgtg gaactgtatt ttgatgcaac cggtcatatt 240

ctgccggatc tgtcaagtct gaaaaccgat ctgcagctga aatacaaaca gaccacccat 300

tttagcgcac gcggctttat gccgtctacc accgcctatc cgtttgtgct gccgaatgca 360

ggcaccgata acgaaaatta tatttttggt cagtgttatt acaaggcaag cgatggcgca 420

ctgtttccgc tggaagtgac cgtgaccctg aataagcgtc tgccggatag tcgtaccagc 480

tatgtgatta cctttctgtg gagcctgaat gcaggcttag ccccggaaac cacccaggcc 540

accttaatta cctctccgtt tacctttagc tatattaccg aagatgat 588

<210> 6

<211> 196

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asn Ser Ile Ala Leu Lys Asn Asn Thr Leu Trp Thr Gly Pro Lys Pro

1 5 10 15

Glu Ala Asn Cys Ile Ile Glu Tyr Gly Lys Glu Asn Pro Asp Ser Lys

20 25 30

Leu Thr Leu Ile Leu Val Lys Asn Gly Gly Ile Val Asn Gly Tyr Val

35 40 45

Thr Leu Met Gly Ala Ser Asp Tyr Val Asn Thr Leu Phe Lys Asn Lys

50 55 60

Asn Val Ser Ile Asn Val Glu Leu Tyr Phe Asp Ala Thr Gly His Ile

65 70 75 80

Leu Pro Asp Leu Ser Ser Leu Lys Thr Asp Leu Gln Leu Lys Tyr Lys

85 90 95

Gln Thr Thr His Phe Ser Ala Arg Gly Phe Met Pro Ser Thr Thr Ala

100 105 110

Tyr Pro Phe Val Leu Pro Asn Ala Gly Thr Asp Asn Glu Asn Tyr Ile

115 120 125

Phe Gly Gln Cys Tyr Tyr Lys Ala Ser Asp Gly Ala Leu Phe Pro Leu

130 135 140

Glu Val Thr Val Thr Leu Asn Lys Arg Leu Pro Asp Ser Arg Thr Ser

145 150 155 160

Tyr Val Ile Thr Phe Leu Trp Ser Leu Asn Ala Gly Leu Ala Pro Glu

165 170 175

Thr Thr Gln Ala Thr Leu Ile Thr Ser Pro Phe Thr Phe Ser Tyr Ile

180 185 190

Thr Glu Asp Asp

195

<210> 7

<211> 576

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggcgcaatta tggccggtaa caaagattat gataaactga ccctgtggac caccccagat 60

ccgtctccga attgtcagat tctggcagaa aatgatgcca aactgaccct gtgtctgacc 120

aaatgtgata gtcagatttt agccaccgtg agcgtgctgg ttgttcgcag cggtaatctg 180

aatccgatta ccggcaccgt gagtagtgca caggtgtttc tgcgctttga tgcaaatggc 240

gtgttactga ccgaacatag caccctgaaa aaatattggg gctatcgtca gggcgatagc 300

attgatggta ctccgtatac caatgccgtt ggctttatgc ctaactcaac cgcctatccg 360

aaaacccagt cttcaaccac caaaaacaac attgtgggcc aggtgtatat gaatggtgac 420

gtgtctaaac cgatgctgtt aaccattacc ttaaacggca cagatgatac cacctcagcc 480

tatagtattt cctttagcta tacctggacc aatggctctt atattggtgc aacctttggc 540

gccaattctt atacctttag ttatattgca caggaa 576

<210> 8

<211> 192

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Gly Ala Ile Met Ala Gly Asn Lys Asp Tyr Asp Lys Leu Thr Leu Trp

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Thr Thr Pro Asp Pro Ser Pro Asn Cys Gln Ile Leu Ala Glu Asn Asp

20 25 30

Ala Lys Leu Thr Leu Cys Leu Thr Lys Cys Asp Ser Gln Ile Leu Ala

35 40 45

Thr Val Ser Val Leu Val Val Arg Ser Gly Asn Leu Asn Pro Ile Thr

50 55 60

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

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Val Leu Leu Thr Glu His Ser Thr Leu Lys Lys Tyr Trp Gly Tyr Arg

85 90 95

Gln Gly Asp Ser Ile Asp Gly Thr Pro Tyr Thr Asn Ala Val Gly Phe

100 105 110

Met Pro Asn Ser Thr Ala Tyr Pro Lys Thr Gln Ser Ser Thr Thr Lys

115 120 125

Asn Asn Ile Val Gly Gln Val Tyr Met Asn Gly Asp Val Ser Lys Pro

130 135 140

Met Leu Leu Thr Ile Thr Leu Asn Gly Thr Asp Asp Thr Thr Ser Ala

145 150 155 160

Tyr Ser Ile Ser Phe Ser Tyr Thr Trp Thr Asn Gly Ser Tyr Ile Gly

165 170 175

Ala Thr Phe Gly Ala Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu

180 185 190

<210> 9

<211> 609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aacaatattt gtattgatga taatattaat accctgtgga ccggcgtgaa tccgacccgt 60

gccaattgtc agattatggc ctctagcgaa agcaatgatt gtaaactgat tctgacctta 120

gttaaaaccg gcgccttagt gaccgcattt gtgtatgtga ttggtgtgtc taatgatttt 180

aatatgctga ccacccataa aaatattaat tttaccgcag aactgttctt tgatagcacc 240

ggcaatctgt taacctcatt atcttctctg aaaaccccgc tgaatcataa atctggtcag 300

aatatggcaa ccggtgcact gaccaatgcc aaaggcttta tgccgagtac caccgcctat 360

ccgtttaatg tcaactctcg cgaaaaagaa aattatatct acggcacctg ttattatacc 420

gcaagcgatc ataccgcatt tccgattgat attagcgtga tgctgaatca gcgcgcctta 480

aacaacgaaa ccagctattg tattcgtgtt acctggagtt ggaataccgg cgttgccccg 540

gaagtgcaga cctcagccac caccctggtt accagtccgt ttacctttta ttatattcgc 600

gaagatgat 609

<210> 10

<211> 203

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Asn Asn Ile Cys Ile Asp Asp Asn Ile Asn Thr Leu Trp Thr Gly Val

1 5 10 15

Asn Pro Thr Arg Ala Asn Cys Gln Ile Met Ala Ser Ser Glu Ser Asn

20 25 30

Asp Cys Lys Leu Ile Leu Thr Leu Val Lys Thr Gly Ala Leu Val Thr

35 40 45

Ala Phe Val Tyr Val Ile Gly Val Ser Asn Asp Phe Asn Met Leu Thr

50 55 60

Thr His Lys Asn Ile Asn Phe Thr Ala Glu Leu Phe Phe Asp Ser Thr

65 70 75 80

Gly Asn Leu Leu Thr Ser Leu Ser Ser Leu Lys Thr Pro Leu Asn His

85 90 95

Lys Ser Gly Gln Asn Met Ala Thr Gly Ala Leu Thr Asn Ala Lys Gly

100 105 110

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Val Asn Ser Arg Glu

115 120 125

Lys Glu Asn Tyr Ile Tyr Gly Thr Cys Tyr Tyr Thr Ala Ser Asp His

130 135 140

Thr Ala Phe Pro Ile Asp Ile Ser Val Met Leu Asn Gln Arg Ala Leu

145 150 155 160

Asn Asn Glu Thr Ser Tyr Cys Ile Arg Val Thr Trp Ser Trp Asn Thr

165 170 175

Gly Val Ala Pro Glu Val Gln Thr Ser Ala Thr Thr Leu Val Thr Ser

180 185 190

Pro Phe Thr Phe Tyr Tyr Ile Arg Glu Asp Asp

195 200

<210> 11

<211> 609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aacaacattt gcattgatga caatattaac accttatgga caggagtcaa ccccaccgaa 60

gccaactgtc aaatcatgaa ctccagtgaa tctaatgatt gcaaattaat tctaacacta 120

gttaaaactg gagcactagt cactgcattt gtttatgtta taggagtatc taacaatttt 180

aatatgctaa ctacacacag aaatataaat tttactgcag agctgttttt cgattctact 240

ggtaatttac taactagact ctcatccctc aaaactccac ttaatcataa atcaggacaa 300

aacatggcta ctggtgccat tactaatgct aaaggtttca tgcccagcac gactgcctat 360

cctttcaatg ataattctag agaaaaagaa aactacattt acggaacttg ttactacaca 420

gctagtgatc gcactgcttt tcccattgac atatctgtca tgcttaaccg aagagcaata 480

aatgacgaga catcatattg tattcgtata acttggtcct ggaacacagg agatgcccca 540

gaggtgcaaa cctctgctac aaccctagtc acctccccat ttacctttta ctacatcaga 600

gaagacgac 609

<210> 12

<211> 203

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Asn Asn Ile Cys Ile Asp Asp Asn Ile Asn Thr Leu Trp Thr Gly Val

1 5 10 15

Asn Pro Thr Glu Ala Asn Cys Gln Ile Met Asn Ser Ser Glu Ser Asn

20 25 30

Asp Cys Lys Leu Ile Leu Thr Leu Val Lys Thr Gly Ala Leu Val Thr

35 40 45

Ala Phe Val Tyr Val Ile Gly Val Ser Asn Asn Phe Asn Met Leu Thr

50 55 60

Thr His Arg Asn Ile Asn Phe Thr Ala Glu Leu Phe Phe Asp Ser Thr

65 70 75 80

Gly Asn Leu Leu Thr Arg Leu Ser Ser Leu Lys Thr Pro Leu Asn His

85 90 95

Lys Ser Gly Gln Asn Met Ala Thr Gly Ala Ile Thr Asn Ala Lys Gly

100 105 110

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Asp Asn Ser Arg Glu

115 120 125

Lys Glu Asn Tyr Ile Tyr Gly Thr Cys Tyr Tyr Thr Ala Ser Asp Arg

130 135 140

Thr Ala Phe Pro Ile Asp Ile Ser Val Met Leu Asn Arg Arg Ala Ile

145 150 155 160

Asn Asp Glu Thr Ser Tyr Cys Ile Arg Ile Thr Trp Ser Trp Asn Thr

165 170 175

Gly Asp Ala Pro Glu Val Gln Thr Ser Ala Thr Thr Leu Val Thr Ser

180 185 190

Pro Phe Thr Phe Tyr Tyr Ile Arg Glu Asp Asp

195 200

<210> 13

<211> 603

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aataacattt gtattgatga taatattaat accctgtgga ccggtgtgaa tccgaccgaa 60

gccaattgtc agatgatgga tagtagcgaa agcaatgatt gtaaactgat tctgacctta 120

gttaaaaccg gcgcactggt gaccgccttt gtgtatgtga ttggcgtttc aaataacttt 180

aatatgttaa ccacctatcg taatattaat tttaccgcag aactgttctt cgatagcgca 240

ggcaatctgc tgacctctct gtctagtctg aaaaccccgc tgaatcataa aagtggtcag 300

aatatggcaa ccggtgccat taccaatgcc aagagcttta tgccgagcac caccgcctat 360

ccgtttaaca acaattcacg cgaaaattat atatacggca cctgtcatta taccgcctca 420

gatcataccg cctttccgat tgatattagt gttatgctga atcagcgtgc cattcgtgca 480

gataccagct attgtattcg cattacctgg agttggaata ccggcgatgc cccggaaggc 540

cagacctctg caaccaccct ggtgaccagt ccgtttacct tttattatat tcgcgaagat 600

gat 603

<210> 14

<211> 201

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Asn Asn Ile Cys Ile Asp Asp Asn Ile Asn Thr Leu Trp Thr Gly Val

1 5 10 15

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

20 25 30

Asp Cys Lys Leu Ile Leu Thr Leu Val Lys Thr Gly Ala Leu Val Thr

35 40 45

Ala Phe Val Tyr Val Ile Gly Val Ser Asn Asn Phe Asn Met Leu Thr

50 55 60

Thr Tyr Arg Asn Ile Asn Phe Thr Ala Glu Leu Phe Phe Asp Ser Ala

65 70 75 80

Gly Asn Leu Leu Thr Ser Leu Ser Ser Leu Lys Thr Pro Leu Asn His

85 90 95

Lys Ser Gly Gln Asn Met Ala Thr Gly Ala Ile Thr Asn Ala Lys Ser

100 105 110

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Asn Asn Ser Arg Glu

115 120 125

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

130 135 140

Phe Pro Ile Asp Ile Ser Val Met Leu Asn Gln Arg Ala Ile Arg Ala

145 150 155 160

Asp Thr Ser Tyr Cys Ile Arg Ile Thr Trp Ser Trp Asn Thr Gly Asp

165 170 175

Ala Pro Glu Gly Gln Thr Ser Ala Thr Thr Leu Val Thr Ser Pro Phe

180 185 190

Thr Phe Tyr Tyr Ile Arg Glu Asp Asp

195 200

<210> 15

<211> 609

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aacaacattt gcattgatga caatattaac accctgtgga caggaattaa ccccaccgaa 60

gccaactgtc aaatgatgga ctccagtgaa tctaatgatt gcaaattaat tctaacacta 120

gttaaaactg gagccctagt cactgcattt gtttatgtta taggagtatc taacaatttt 180

aatatgctaa ctacatacag aaatataaat tttactgcgg agctgttttt tgattctgcg 240

ggtaatttac taactagcct gtcatcccta aaaactccac ttaatcataa atcaggacaa 300

aacatggcta ctggtgccat tactaatgct aaaagtttca tgcccagcac aactgcttat 360

cctttcaata ataattctag agaaaaagaa aactacattt acggaacctg tcactacaca 420

gctagtgatc acactgcttt tcccattgac atatctgtca tgcttaacca aagagcaata 480

agagctgata catcatattg tattcgtata acttggtcct ggaacacagg agatgcccca 540

gaggggcaaa cctctgctac aaccctagtt acctccccat ttacctttta ctacatcaga 600

gaagacgac 609

<210> 16

<211> 203

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Asn Asn Ile Cys Ile Asp Asp Asn Ile Asn Thr Leu Trp Thr Gly Ile

1 5 10 15

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

20 25 30

Asp Cys Lys Leu Ile Leu Thr Leu Val Lys Thr Gly Ala Leu Val Thr

35 40 45

Ala Phe Val Tyr Val Ile Gly Val Ser Asn Asn Phe Asn Met Leu Thr

50 55 60

Thr Tyr Arg Asn Ile Asn Phe Thr Ala Glu Leu Phe Phe Asp Ser Ala

65 70 75 80

Gly Asn Leu Leu Thr Ser Leu Ser Ser Leu Lys Thr Pro Leu Asn His

85 90 95

Lys Ser Gly Gln Asn Met Ala Thr Gly Ala Ile Thr Asn Ala Lys Ser

100 105 110

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Asn Asn Ser Arg Glu

115 120 125

Lys Glu Asn Tyr Ile Tyr Gly Thr Cys His Tyr Thr Ala Ser Asp His

130 135 140

Thr Ala Phe Pro Ile Asp Ile Ser Val Met Leu Asn Gln Arg Ala Ile

145 150 155 160

Arg Ala Asp Thr Ser Tyr Cys Ile Arg Ile Thr Trp Ser Trp Asn Thr

165 170 175

Gly Asp Ala Pro Glu Gly Gln Thr Ser Ala Thr Thr Leu Val Thr Ser

180 185 190

Pro Phe Thr Phe Tyr Tyr Ile Arg Glu Asp Asp

195 200

Claims (7)

1. A hybridoma cell capable of secreting an anti-Ad3FK monoclonal antibody is characterized in that the preservation number is CCTCC No: C201875.

2. the anti-Ad3FK monoclonal antibody secreted by the hybridoma cell of claim 1, wherein the anti-Ad3FK monoclonal antibody is labeled as 1B 5.

3. An anti-Ad3FK monoclonal antibody, wherein the light chain of the anti-Ad3FK monoclonal antibody comprises the amino acid sequence shown as SEQ ID No.1, and the heavy chain of the anti-Ad3FK monoclonal antibody comprises the amino acid sequence shown as SEQ ID No. 2.

4. Use of a hybridoma cell according to claim 1 or an anti-Ad3FK monoclonal antibody according to any one of claims 2 to 3 for the preparation of a medicament for the treatment of a disease associated with infection with a human adenovirus type 3.

5. A medicament for treating adenovirus 3-associated diseases in humans, comprising the hybridoma cell of claim 1 or the anti-Ad3FK monoclonal antibody of any one of claims 2 to 3.

6. The medicament for treating human type 3 adenovirus-associated disease according to claim 5, further comprising pharmaceutically acceptable excipients.

7. A detection reagent comprising the anti-Ad3FK monoclonal antibody of any one of claims 2 to 3.

Images