CN112159467A - Antibodies capable of binding to AAV1-13 - Google Patents

Abstract

The invention relates to the field of immunology, in particular to an antibody capable of binding to AAV 1-13. The antibody can recognize VP1, VP2 and VP3 proteins of all serotypes of AAV1-13, has high affinity and good specificity, and can be more conveniently used for separating and identifying adeno-associated viruses because the VP proteins are positioned on the surfaces of the adeno-associated viruses.

Description

Antibodies capable of binding to AAV1-13

Technical Field

The invention relates to the field of immunology, in particular to an antibody capable of binding to AAV 1-13.

Background

Adeno-associated Virus (AAV) belongs to the parvoviridae (parvoviridae), and is an icosahedral parvovirus which cannot autonomously replicate and has no envelope, the diameter of which is about 20-26nm, and the structure of single-stranded linear DNA. Modified rAAV viral tools have been widely used for gene expression, gene manipulation, and gene therapy at the animal level. At present, 13 serotypes of AAV (namely AAV1-13) exist, and different serotypes have different affinities to tissues or organs, wherein AAV2, AAV3 and AAV9 are derived from human beings and are adeno-associated virus vectors which are researched most thoroughly and applied most widely to date. AAV is a simple, non-pathogenic, single-stranded DNA virus that requires a helper virus, usually an adenovirus (Ad) or a Herpes Simplex Virus (HSV), to participate in the life cycle. The genome terminates with Inverted Terminal Repeats (ITRs), and the intermediate genome encodes two proteins: cap and Rep. The Cap gene encodes three capsid proteins, VP1, VP2, and VP 3.

Purification of AAV currently uses iodixanol density gradient centrifugation, and if a purification method using ion exchange chromatography including reverse ion exchange chromatography (including cation and anion chromatography) is used, although the purity is high, the recovery efficiency is low, and the conditions are severe.

Disclosure of Invention

The invention relates to an antibody, which comprises a heavy chain complementary determining region H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequences of the heavy chain complementary determining region H-CDR1, H-CDR2 and H-CDR3 are sequentially shown as SEQ ID NO. 1-3, and a light chain complementary determining region L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequences of the light chain complementary determining region L-CDR1, L-CDR2 and L-CDR3 are sequentially shown.

The invention also relates to nucleic acids, vectors and host cells for expression production of the antibodies.

The invention also relates to a method for producing said antibodies.

The invention also relates to a reagent, a test strip or a kit for isolating or identifying adeno-associated virus or adeno-associated virus fragments, which comprises the antibody.

The invention also relates to a chromatographic medium for isolating adeno-associated virus or adeno-associated virus fragments, comprising a matrix support and an antibody as described above immobilized on the matrix support.

The invention has the beneficial effects that:

can recognize VP proteins (VP1, VP2 and VP3 proteins) of all serotypes of AAV1-13, has high affinity and good specificity, and can be more conveniently used for separating and identifying adeno-associated virus because the VP proteins are positioned on the surface of the adeno-associated virus.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a diagram of an antigen-containing expression vector constructed in one embodiment of the present invention;

FIG. 2 is an electrophoretogram following expression of an antigenic protein in accordance with an embodiment of the present invention;

FIG. 3 is an electrophoretogram of an antigen protein after purification according to an embodiment of the present invention; lanes 1 and 2 are lanes in which the band of the target protein is located;

FIG. 4 is a graph showing the result of Western Blot detection of the obtained antibody in one example of the present invention; wherein FIG. 4A is an antibody claimed in the present invention, and FIGS. 4B to 4E are control antibodies; the lanes of each electrophoretogram are sequentially Marker, AAV 1-13;

FIG. 5 shows the titer of the antibody obtained by ELISA in one embodiment of the present invention;

FIG. 6 is a graph showing the validation of the application of affinity chromatography to the resulting antibody in one embodiment of the present invention; wherein, FIG. 6A shows the electrophoresis results after elution of AAV 1-AAV 6; FIG. 6B shows the results of the electrophoresis of AAV 7-AAV 13 after elution.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to an antibody, which comprises a heavy chain complementary determining region H-CDR1, H-CDR2 and H-CDR3, wherein the amino acid sequences of the heavy chain complementary determining region H-CDR1, H-CDR2 and H-CDR3 are sequentially shown as SEQ ID NO. 1-3, and a light chain complementary determining region L-CDR1, L-CDR2 and L-CDR3, wherein the amino acid sequences of the light chain complementary determining region L-CDR1, L-CDR2 and L-CDR3 are sequentially shown.

The antibody can specifically recognize the overlapping regions of VP1, VP2 and VP3, and can recognize any one or more (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13) AAV1-13, so that the antibody can be widely used for AAV enrichment/detection, and is particularly suitable for the case that the serotype of AAV in a sample to be treated is unknown or contains multiple AAV serotypes.

In the present invention, the term "antibody" is a protein that binds to a specific antigen, and broadly refers to all proteins and protein fragments that include complementarity determining regions (CDR regions), particularly full-length antibodies or functional fragments of antibodies. The term "full-length antibody" includes both polyclonal and monoclonal antibodies, and the term "antibody functional fragment" is a substance that comprises part or all of the CDRs of an antibody, which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to an antigen. Such fragments are biologically active in that they bind to a target antigen and can compete with other antigen binding molecules (including whole antibodies) for binding to a given epitope. In some embodiments, the antibody or functional fragment of an antibody has the effect of specifically recognizing and binding any one of AAV 1-13. In some embodiments, the antibody or functional fragment of an antibody binds to a VP protein fragment of any one of AAV 1-13. This binding is usually specific. In one aspect, such fragments will comprise a single heavy chain and a single light chain, or portions thereof. Such fragments may be produced by recombinant nucleic acid techniques, or may be produced by enzymatic or chemical cleavage of antigen binding molecules, including intact antibodies.

The term "complementarity determining regions" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al (Kabat et al, Sequences of proteins of immunological interest,5th Ed, "US Department of Health and Human Services, NIH,1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, the term "CDR" is used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to the antigen or epitope it recognizes, depending on the situation. In another embodiment, the CDR regions or CDRs refer to the highly variable regions of the heavy and light chains of an immunoglobulin as defined by IMGT.

The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody is administered at a rate of about less than 10^-6M, e.g. less than about 10^-7M、10^{- 8}M、10^-9M or 10^-10M or less affinity (K)_D) And (4) combining.

Variants of the antibodies are also within the scope of the claimed invention, for example sequences each having at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than 99% identity to the respective CDR or FR, or variable region VL and/or VH, or the full length amino acid or nucleotide sequence of the antibody described herein. In some cases, a variant of an antibody comprises at least the 6 CDRs described above; in some cases, a variant of an antibody comprises at least one heavy chain and one light chain, while in other cases, the variant form contains two identical light chains and two identical heavy chains (or subparts thereof). In some cases, antibody variants are conservative modifications or conservative substitutions or substitutions of antibody sequences provided herein. "conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) so that changes can be made frequently without changing the biological activity of the protein. It is known to The person skilled in The art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter The biological activity (see, for example, Watson et al (1987) Molecular Biology of The Gene, The Benjamin/Cummings pub. Co., p. 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to abolish biological activity.

In some embodiments, the antibody further comprises a heavy chain framework region H-FR1, H-FR2, H-FR3 and H-FR4 having the sequence shown in SEQ ID NO 7-10; and/or; the sequences are shown as SEQ ID NO 11-14 in sequence, namely a light chain framework region L-FR1, L-FR2, L-FR3 and L-FR 4.

In some embodiments, the antibody has a constant region and the heavy chain constant region sequence is selected from the constant region sequences of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD; the light chain constant region is a kappa or lambda chain.

In some embodiments, the species source of the constant region is selected from cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fighting, or human.

The invention also relates to nucleic acids encoding the antibodies as described above.

The nucleic acid is typically RNA or DNA, and the nucleic acid typically carries a signal peptide sequence given that antibodies are membrane proteins.

The invention also relates to a vector comprising a nucleic acid as described above.

The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.

The invention also provides a cell comprising a nucleic acid as described above or a vector as described above.

Preferred host cells suitable for expressing the antibodies of the invention include: mammalian cells such as NS0, Sp2/0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells may be used, thus allowing the molecule to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines may be employed.

The present invention also provides a method of producing an antibody as described above, comprising:

culturing the cells as described above under suitable culture conditions; and

the antibody thus produced is recovered from the culture medium or from the cultured cells.

Suitable culture conditions may be a commonly used medium suitable for the cell culture, and the antibody is isolated from the culture medium supernatant;

alternatively, the cells are transferred to an animal (preferably a nude mouse), and the antibody is collected from ascites, serum or spleen of the animal.

According to a further aspect of the invention, the invention also relates to a reagent, a test strip or a kit for isolating or identifying an adeno-associated virus or an adeno-associated virus fragment, comprising an antibody as described above.

The invention also relates to a chromatographic medium for separating the adeno-associated virus or adeno-associated virus fragment, which comprises a matrix support and the antibody fixed on the matrix support, wherein the antibody is the antibody.

In some embodiments, the matrix support comprises any one of agar, agarose derivatives, magnetic beads, silica, titanium dioxide, alginate, cellulose derivatives, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters, gelatin, silicon, ceramics, glass (e.g., borosilicate glass), polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone, or copolymers formed of any of several.

The substrate support may be of any shape, e.g., substantially spherical, substantially cubic, etc.

In some embodiments, the matrix support comprises Sepharose 4Fast Flow or CNBr-Activated Sepharose 4Fast Flow.

In a preferred embodiment, the matrix support is porous.

The invention also relates to a chromatographic separation device containing a chromatographic medium as described above.

There are many types of chromatographic separation devices, including but not limited to the following: SPE solid phase extraction column, centrifuge tube with separation membrane, magnetic bead, separation membrane (membranes), fast detection biochip (bio-chips), fiber bundle column, chromatographic separation device is preferably column, such as monolithic column and conventional analytical grade or preparative grade chromatographic column.

According to a further aspect of the invention, the invention also relates to the use of an antibody as described above, or a chromatographic medium as described above, or a chromatographic separation device as described above, for removing adeno-associated virus or adeno-associated virus fragments from a liquid phase composition;

the adeno-associated virus is selected from any 1 or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 serotypes of AAV 1-13.

In some embodiments, the liquid composition is a cell or tissue lysate, serum, ascites fluid, or cell culture supernatant.

Embodiments of the present invention will be described in detail with reference to examples.

EXAMPLE 1 preparation of antibodies

By taking VP1 of AAV8 as a basis, AAV1-13 is compared, most of non-homologous sequences are removed to obtain a VPx sequence, the VPx sequence is constructed on a pET-21 vector, and the representation is expressed and purified to be used as an antigen to obtain a mouse monoclonal antibody.

1. Construction of antigen overexpression prokaryotic vector

(1) Plasmid pET-21(a) + -P24 was digested simultaneously with Nde1 and Xho I. 50 mu L of enzyme digestion reaction system contains pET-21(a) + -P2410 ug, 10 XCutsmart (NEB)5 mu L, Nde 11 mu L Xho I1 mu L, water is used for complementing to 50 mu L, 1% agarose gel electrophoresis is carried out after enzyme digestion is carried out for 4h at 37 ℃, a large segment is cut by a blade under an ultraviolet lamp, and recovery and purification are carried out;

(2) synthesizing a VPx sequence, adding Nde1 enzyme cutting sites and Xho I enzyme cutting sites at the upstream and the downstream respectively, and constructing the VPx sequence into a puc19 vector, wherein the vector is named as puc 19-VPx;

(3) plasmid puc19-VPx was double digested with Nde1 and Xho I: 50 mu L of enzyme digestion reaction system contains puc19-VP 810 ug, 10 XCutsmart (NEB)5 mu L, BamHI 1 mu L, Hind III 1 mu L is complemented to 50 mu L with water, 1% agarose gel electrophoresis is carried out after enzyme digestion is carried out for 4h at 37 ℃, a 1.7kb fragment is cut by a blade under an ultraviolet lamp and is recovered and purified;

(4) 1 mu L of enzyme-digested pET-21(a) + -P24 vector, 3 mu L of fragment VPx, 1 mu L of enzyme and 1 mu L of buffer, supplementing water to 10 mu L, connecting at 16 ℃ for 1h, and then placing on ice for conversion plating;

(5) single colonies were picked and identified by plasmid mininote sequencing. The expression vector (figure 1) was successfully constructed by sequencing.

2. Recombinant protein expression screening

2.1 plasmid transformation

2.2 inoculation

2.3 expression culture: transferring the overnight cultured seed solution into 5ml LB culture at 37 deg.C and 250rpm to OD according to a ratio of 1:100(V: V)₆₀₀When the concentration reaches 0.8, adding IPTG with the final concentration of 0.5mM for induction, and respectively continuing to culture at 37 ℃ for 3h or culturing at 16 ℃ overnight;

2.4 ultrasonic disruption: respectively collecting induced bacteria, resuspending the collected bacteria in an ice-bath lysine Buffer (50 mM Tris-HCl,300mM NaCl, 10% Glycerol, 1mM PMSF, 0.1% TritonX-100) according to a ratio of 1:20(g/V), carrying out ultrasonic disruption (super 2s, 3s, ultrasonic 1min), and carrying out 12000rmp centrifugation to collect supernatant and precipitate after ultrasonic disruption;

2.5 expression detection: expression was detected on a 15% SDS-PAGE gel; the results are shown in FIG. 2.

The samples in FIG. 2 correspond to four sets of single colony tests (each set being an uninduced control and an induced set).

3 amplifying expression and purification

3.1 high-level expression;

3.2 ultrasonic crushing, centrifuging and collecting ultrasonic supernatant (ultrasonic conditions: over 5s, within 10s, ultrasonic treatment for 30min in total, ice bath);

3.3 column loading: slowly adding the ultrasonic supernatant to a pre-treated 5ml Ni-NTA column according to 1ml/min, and collecting the upper column flow-through;

3.4 column equilibration: after the sample was applied, the sample was washed with 5CV of Buffer 2: column equilibration 20mM PB,300mM NaCl, 5% Glycerol pH 7.4;

3.5 gradient elution: eluting with Buffer 3, Buffer4 and Buffer5 in sequence, and collecting elution peaks:

Buffer3：20mM PB,300mM NaCl,5％Glycerol，20mM Imidazole pH7.4；

Buffer4：20mM PB,300mM NaCl,5％Glycerol，50mM Imidazole pH7.4；

Buffer5：20mM PB,300mM NaCl,5％Glycerol，200mM Imidazole pH7.4；

3.6 purity detection: performing purity detection on 15% SDS-PAGE, and combining samples with the same purity;

3.7 concentration detection: protein concentration detection was performed by BCA method.

4. Two-step dialysis

After combining the target proteins eluted from the Ni-NTA column by 200mM imidazole, dialyzing the combined target proteins against pre-cooled 20mM PB,300mM NaCl and 5% Glycerol at a ratio of 1:100(V: V), and replacing the dialysate every 2h for three times in total; then continuously transferring the sample dialyzed in the last step into precooled PBS, continuously dialyzing at pH7.4, replacing the dialysate every 2h for 3 times in total, and finally dialyzing overnight for the last time; the electrophoresis results of the dialyzed protein are shown in FIG. 3.

5. Preparation of monoclonal antibodies

5.1 mouse immunization:

immunization of BALB/c mice immunization was performed according to a conventional method, 200. mu.L of blood was collected by a tail-off vacuum method for antibody detection one week after the second immunization, and the mice were allowed to rest for two weeks for boosting immunization after the second immunization blood collection, and fused after three days.

5.2 cell fusion:

cell fusion was carried out using 50% PEG of molecular weight 1450 as a fusogenic agent, and was carried out according to the conventional method, which was carried out as follows:

take 1X 10⁷The SP2/0 myeloma cells were mixed with the immune spleen cells, resuspended in 15mL of RPMI-1640 basic medium, and washed twice at 1500r/min for 5 min. The sterilized absorbent paper was removed from the clean bench, and the supernatant of a 50mL centrifuge tube containing the mixture of myeloma cells and spleen cells was poured off, and the tube was inverted on the absorbent paper to remove water droplets. Using a small aluminum pot to contain 2/3 volumes of water, heating the water on an electric furnace to 37 ℃, and then putting the water into a superclean bench; from CO₂Taking out 50% PEG incubated at 37 ℃ from the incubator, sucking 0.8mL by using a 1mL suction tube, holding a 50mL centrifuge tube filled with mixed cells by hand, placing the centrifuge tube in a small aluminum pot with a water bath at 37 ℃, slowly adding PEG to the mixed cells while gently stirring, continuing for 90Sec, and then centrifuging the centrifuge tubeInserting into a centrifuge tube rack, removing small aluminum pot, and removing CO₂The incubator takes out 50mL RPMI-1640 basic culture solution incubated at 37 ℃, sucks 10mL by a pipette, slowly adds the solution to the fused cells while gently stirring to disperse cell masses, firstly adds 1mL, then adds 2mL, then adds 3mL, finally adds the rest 4mL, after adding the first 10mL, then adds the rest 40mL along the tube wall, after adding, screws the cover, and repeatedly reverses for several times to mix the cells. 1500r/min, centrifuge for 5min, discard the supernatant, resuspend the fused cells in 72mL RPMI-1640 complete medium with 1% HAT in which the feeder cells were resuspended. Note: the action should be light, and the cells will be gently stirred up with a pipette without blowing. The resuspended cells were added dropwise to a 96-well cell culture plate at 2 drops/well and CO was added₂Culturing in an incubator. Microcolonies were observed on day 4 post-fusion; on day 7, 1 drop/well of RPMI-1640 complete medium containing 1% HT was added; on about day 10, colonies grew to 1/4% and the culture medium turned yellow, and antibody detection was performed.

5.3 screening and cloning of hybridoma Positive clones:

on day 7 after fusion, the plates were observed under an inverted microscope, and a small dot was marked with a marker over the plate lid with the well where the colony appeared, to mark the position of the colony for sampling and registration of the original well during testing.

Culture wells with hybridoma cell growth were screened by indirect ELISA.

5.4 mouse ascites method monoclonal antibody production:

and (3) enlarging culture of positive holes: feeder cells were added to 24-well cell culture plates, 4 drops/well, and positive cells in 96-well plates were transferred to 24-well cell culture plates for expanded culture.

And (3) injection: after the cells grow to the bottom of the full-length hole, resuspending the cells in a 24-hole plate, centrifuging at 1200r/min for 5min, counting the cells, and diluting the cells to 1 × 10 by using RPMI-1640 basic culture solution⁶～5×10⁶Each mouse is injected into the abdominal cavity by 0.5mL, and the abdominal bulge of the mouse is observed about 8 days, so that the ascites can be collected.

EXAMPLE 2 detection of antibodies

The antibody prepared in example 1 was screened to obtain VP-18 antibody, and the amino acid sequences of the heavy chain variable region and the light chain variable region were shown in SEQ ID NO 15 and 16, respectively, by sequencing.

Detection of antibody by Western Blot

Sample preparation

Taking 15 mu L of AAV1-13 purified virus, adding loading buffer, adding according to 1:4(loading buffer: cell sap), mixing, boiling at 95 deg.C for 5min, placing on a dry ice box, then centrifuging at 4 deg.C 12000g for 5 min.

Preparation of SDS-PAGE gels: 10% of the separation gum and 5% of the concentration gum were used.

SDS-PAGE gel electrophoresis

a. Fixing the gel plate in the inner groove of the electrophoresis apparatus, placing the fixed gel plate in the outer groove, and adding a proper amount of 1 xSDS electrophoresis buffer solution into the outer groove. Adding electrophoresis buffer solution into the inner tank until the gel sample adding hole is just submerged;

b. carefully pull out the comb to avoid tearing the polyacrylamide gel sample application hole. After the comb is pulled out, washing and filling the sample adding hole with 1 xSDS electrophoresis buffer;

c. the samples were loaded and subjected to SDS-PAGE electrophoresis. Connecting a power supply, firstly carrying out electrophoresis at the voltage of 80V for 30min until bromophenol blue dye enters separation gel from concentrated gel, then regulating the voltage to 120V, and continuing electrophoresis for 70min until bromophenol blue reaches the bottom of the gel;

d. the power is turned off and the connected wires are removed, the running buffer is discarded, and the gel sandwich is taken out.

Rotary film

a. Before the completion of electrophoresis, 1 PVDF membrane and 6 filter papers having the same size as the gel were prepared. Activating the PVDF membrane by methanol for 5min, and then balancing in a buffer solution for 15 min; the filter paper is balanced in the transfer buffer for 15 min;

b. a clamp for transferring the membrane, two sponge pads, filter paper and a soaked membrane are placed in a tray with transfer liquid;

c. the clamp is opened to keep the black side horizontal. A spongy cushion is arranged on the upper surface. Filling three layers of filter paper on the cushion to remove air bubbles in the filter paper;

d. prying off the glass plate, gently scraping off the concentrated gel, and gelling in ddH₂Wash in O and then in 1 Xspin-membrane buffer. The release gel was carefully applied to the filter paper and manually aligned with the filter paper. The film was covered on the glue, filled with the whole glue and degassed of bubbles. The membrane was covered with 3 sheets of filter paper and the air bubbles were removed. Finally, covering another spongy cushion, and closing the clamp;

e. the clip is placed in the transfer tank so that the black side of the clip faces the black side of the tank and the white side of the clip faces the red side of the tank. During the electric transfer, heat is generated, and ice is put on one side of the groove to reduce the temperature. Typically, the transfer is carried out with 300mA for 2 h.

Immune response

a. Transferring the membrane into a TBST incubation box containing 5% skimmed milk powder, and shaking and sealing on a shaking table at room temperature for 1 h;

b. diluting primary antibody with TBST containing 5% skimmed milk powder to appropriate concentration; taking out the membrane from the confining liquid, placing the protein surface of the membrane on the liquid surface of the antibody upwards, and lifting four corners of the membrane to remove residual bubbles; shaking and incubating overnight at 4 ℃ by a decolorizing shaker;

c. washing with TBST at room temperature for 10min three times on a decolorizing shaker;

d. and (3) diluting the secondary antibody (anti-mouse) by the same method, contacting the secondary antibody with the membrane, incubating for 1-2 h at room temperature, washing for three times by a TBST (tert-butyl-.

Chemiluminescence

Mixing the reagent A and the reagent B in equal volume; and (3) putting the white board with the protein surface of the film facing upwards, dripping the luminous liquid on the film to fully cover the film surface, and then putting the film into an imager for photographing.

Because the antigen epitope in the recombinant protein antigen fragment is more, through screening, the applicant obtains 40 monoclonal antibodies which can be combined with various adeno-associated viruses, the WB result is shown in figure 4, the antibody result which has the best effect and is claimed by the invention is shown in figure 4A, and the antibody can be combined with AAV 1-13; the results of the partial cell lines are shown in FIGS. 4B to 4E.

ELISA for detecting antibody titer of VP-18 antibody to different serotype AAV

The method comprises the following steps:

1. recombinant AAV (serotypes 1-13) was diluted to 5X 10 with coating buffer (pH9.6,0.05mol/L carbonate buffer)⁹vp/ml, 100 mu L/well spreading an enzyme label plate, and 37 ℃ for 1 hour;

2. the supernatant was discarded and washed three times with 100. mu.L of washing buffer (PBS);

3.100. mu.L, 10% mil, coating buffer, blocking at 37 ℃ for 1 hour;

4. washing was repeated 3 times;

5. cell culture supernatant, 100. mu.L/WELL, 1 hour at 37 ℃;

6. washing three times with washing buffer (PBS);

HRP secondary antibody, diluted as described, added 1% BSA with PBS, 100 μ Ι,1 hour at 37 degrees;

8. washing three times with washing buffer (PBS);

9. adding a chromogenic substrate TMB;

stopping the reaction with 1M sulfuric acid at 10.37 ℃ for 15-30 minutes;

11. detection OD of enzyme-linked immunosorbent assay (OD)₄₅₀Light is absorbed.

The results of the detection are shown in FIG. 5. It can be seen from the figure that the VP-18 antibody has higher antibody titer for different serotypes of AAV.

Application of VP-18 antibody in affinity chromatography

1. Antibody coupling and activating filler step

The affinity filler was prepared by coupling AAV antibodies to Sepharose 4Fast Flow, with reference to the manual of use of NHS-activated Sepharose 4Fast Flow.

2. Step of affinity purification of Filler for AAV

1) Balancing: the loaded column is equilibrated with 5-10 CV of 10mM Tris-HCl,150mM NaCl, pH7.5 solution.

2) Loading: after freezing-thawing and cracking HEK293 cells respectively packaged with AAV1-13 and centrifuging, filtering by 0.22 mu m, and then loading to a well-balanced chromatographic column, wherein the loading capacity of the loading is not more than 1E +14vg/mL filler.

3) Washing 1: after the loading, the sample is equilibrated with 5-10 CV of 10mM Tris-HCl,150mM NaCl, pH7.5 solution.

4) And (3) washing 2: 5-10 CV of 10mM Tris-HCl,1M NaCl, pH7.5 solution is used to wash the impurities.

5) And (3) washing 2: the column is equilibrated again with 5-10 CV of 10mM Tris-HCl,150mM NaCl, pH7.5 solution.

6) And (3) elution: the column was eluted with 100mM citric acid, pH3.5 and samples were collected after peaking and immediately adjusted to neutral pH with 1M Tris solution.

7) Regeneration: the column was regenerated using 1M citric acid, pH1.5 solution and the residual impurities were washed off the column.

8) Rebalancing: the column is equilibrated with 5-10 CV of 10mM Tris-HCl,150mM NaCl, pH7.5 solution.

9) And (3) storage: and preserving the chromatographic column by using a 20% ethanol solution with 5-10 CV.

3. And (3) purification result:

the eluted samples were subjected to SDS-PAGE followed by Coomassie blue staining, and the results are shown in FIGS. 6A and 6B. As can be seen, the VP-18 antibody has an enrichment effect on different serotype AAV, the carrying capacity is ideal, and the VP-18 antibody hardly generates non-specific combination with other components of HEK293 cell lysate, so that the antibody specificity is good.

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> and Yuan Biotechnology (Shanghai) Ltd

<120> antibody capable of binding to AAV1-13

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213> artificial sequence

<400> 1

Gly Phe Thr Phe Thr Ser Tyr Thr

1 5

<210> 2

<211> 7

<212> PRT

<213> artificial sequence

<400> 2

Ile Asn Val Gly Asp Asp Thr

1 5

<210> 3

<211> 13

<212> PRT

<213> artificial sequence

<400> 3

Ala Arg Gly Arg Asp Gly Phe Tyr Val Val Phe Ala Tyr

1 5 10

<210> 4

<211> 6

<212> PRT

<213> artificial sequence

<400> 4

Gln Ser Ile Ser Asn Asn

1 5

<210> 5

<211> 3

<212> PRT

<213> artificial sequence

<400> 5

Tyr Thr Ser

1

<210> 6

<211> 9

<212> PRT

<213> artificial sequence

<400> 6

Gln Gln Ser Tyr Ser Trp Pro Trp Thr

1 5

<210> 7

<211> 25

<212> PRT

<213> artificial sequence

<400> 7

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser

20 25

<210> 8

<211> 17

<212> PRT

<213> artificial sequence

<400> 8

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

1 5 10 15

Ser

<210> 9

<211> 38

<212> PRT

<213> artificial sequence

<400> 9

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

1 5 10 15

Ala Arg Asn Ile Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser Glu Asp

20 25 30

Thr Ala Ile Tyr Tyr Cys

35

<210> 10

<211> 11

<212> PRT

<213> artificial sequence

<400> 10

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

1 5 10

<210> 11

<211> 26

<212> PRT

<213> artificial sequence

<400> 11

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser

20 25

<210> 12

<211> 17

<212> PRT

<213> artificial sequence

<400> 12

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

1 5 10 15

Lys

<210> 13

<211> 36

<212> PRT

<213> artificial sequence

<400> 13

Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

1 5 10 15

Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr Glu Asp Phe Gly

20 25 30

Met Tyr Phe Cys

35

<210> 14

<211> 10

<212> PRT

<213> artificial sequence

<400> 14

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 10

<210> 15

<211> 119

<212> PRT

<213> artificial sequence

<400> 15

Glu Val Lys Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

Ala Ser Ile Asn Val Gly Asp Asp Thr Ser Tyr Ser Asp Asn Leu Lys

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 16

<211> 107

<212> PRT

<213> artificial sequence

<400> 16

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Tyr Ser Trp Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

Claims (13)

1. The antibody is characterized by comprising a heavy chain complementary determining region H-CDR1, H-CDR2 and H-CDR3 of which the amino acid sequences are shown as SEQ ID NO. 1-3 in sequence, and a light chain complementary determining region L-CDR1, L-CDR2 and L-CDR3 of which the amino acid sequences are shown as SEQ ID NO. 4-6 in sequence.

2. The antibody of claim 1, further comprising H-FR1, H-FR2, H-FR3 and H-FR4 of heavy chain framework regions having the sequence shown in SEQ ID Nos. 7-10; and/or; the sequences are shown as SEQ ID NO 11-14 in sequence, namely a light chain framework region L-FR1, L-FR2, L-FR3 and L-FR 4.

3. The antibody of claim 1 or 2, wherein the antibody has a constant region and the heavy chain constant region sequence is selected from the group consisting of constant region sequences of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD; the light chain constant region is a kappa or lambda chain.

4. The antibody of claim 3, wherein the species of said constant region is derived from a species selected from the group consisting of bovine, equine, bovine, porcine, ovine, caprine, rat, mouse, canine, feline, rabbit, camel, donkey, deer, mink, chicken, duck, goose, turkey, chicken fountains, and human.

5. A nucleic acid encoding the antibody of any one of claims 1 to 4.

6. A vector comprising the nucleic acid of claim 5.

7. A cell comprising the nucleic acid of claim 5 or the vector of claim 6.

8. A method of producing the antibody of any one of claims 1 to 4, comprising:

culturing the cell of claim 7 under suitable culture conditions; and

the antibody thus produced is recovered from the culture medium or from the cultured cells.

9. A reagent, a test strip or a kit for isolating or identifying an adeno-associated virus or an adeno-associated virus fragment, comprising the antibody according to any one of claims 1 to 4.

10. A chromatographic medium for isolating adeno-associated virus or adeno-associated virus fragments, wherein the chromatographic medium comprises a substrate support and an antibody immobilized on the substrate support, wherein the antibody is the antibody according to any one of claims 1 to 4.

11. The chromatography media of claim 10, wherein the matrix support comprises any one of agar, agarose, magnetic beads, silica, titanium dioxide, alginate, cellulose, dextran, starch, cyclodextrin, chitosan, carrageenan, guar gum, gum arabic, gum ghatti, gum tragacanth, karaya gum, locust bean gum, xanthan gum, pectin, mucin, liver thioesters and gelatin, silicon, ceramics, glass, polyurethane, polystyrene divinyl benzene, polymethyl methacrylate, polyacrylamide, polyethylene glycol terephthalate, polyvinyl acetate, polyethylene, polypropylene, polyvinyl chloride, polyvinyl pyrrolidone, or copolymers formed of any of the foregoing.

12. A chromatographic separation device comprising the chromatographic medium according to claim 10 or 11.

13. Use of the antibody of any one of claims 1 to 4, or the chromatographic medium of claim 10 or 11, or the chromatographic separation device of claim 12 for removing adeno-associated virus from a liquid phase composition;

the adeno-associated virus is selected from any 1 or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 serotypes of AAV 1-13.

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