CN114773460A

CN114773460A - Nano antibody of targeted rotavirus protein and application thereof

Info

Publication number: CN114773460A
Application number: CN202210703016.3A
Authority: CN
Inventors: 叶其壮; 白晓康; 王梓杨; 陈锦德; 肖利群
Original assignee: Guangzhou Ming Pharmaceutical Technology Co ltd
Current assignee: Guangzhou Ming Pharmaceutical Technology Co ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-07-22
Anticipated expiration: 2042-06-21
Also published as: CN114773460B

Abstract

Rotavirus is a virus that causes acute gastrointestinal diseases (diarrhea). The invention discloses a nano antibody of a targeted rotavirus capsid protein, and expression, purification and identification thereof. The nano antibody targeting rotavirus can be used for environmental monitoring of rotavirus and clinical diagnosis and treatment of rotavirus infection.

Description

Nano antibody of targeted rotavirus protein and application thereof

Technical Field

The invention relates to the field of antibody technology and biomedicine, in particular to the monitoring of rotavirus transmission and the diagnosis and treatment of rotavirus infected diseases.

Background

Rotavirus (RV) is a double-stranded ribonucleic acid virus, first discovered by Australian scholars in 1973 using an electron microscope, and is named because it resembles a wheel. It is a major cause of severe diarrhea in children worldwide, and in addition it can cause acute gastrointestinal diseases in humans or animals. However, no specific medicine capable of effectively treating rotavirus infection exists in the market so far, so that the research on a rapid and accurate early detection method of rotavirus has profound significance for preventing and treating the virus.

Monoclonal antibodies are mainly prepared by taking experimental mice as hosts, and the preparation method is generally as follows: after animal cells are infected by rotavirus, culture solution obtained by culturing the cells contains more rotavirus, the rotavirus is used for carrying out immune treatment on experimental mice, the mice which are successfully immunized are killed after the treatment, the spleen of the mouse is taken out under the aseptic condition, the spleen and myeloma cells are fused, and positive clones are screened out by methods such as ELISA and the like. Cloning the screened cells to form stable cell strain, culturing the cell strain in vitro or inducing ascites in mouse, purifying from culture medium or ascites to obtain rotavirus monoclonal antibody, and final protein blotting and other steps.

The rotavirus specific antibody prepared by the means at present is a traditional monoclonal antibody, and the traditional monoclonal antibody has the following defects: the preparation method has the advantages of long research and development period, long production period, high preparation cost, high immunogenicity, difficult removal in a body and weak penetration capacity. The nano antibody derived from alpaca and other animals keeps the affinity and specificity of the traditional monoclonal antibody and has the following advantages: the expression quantity in prokaryotic or eukaryotic cells is high, the growth cycle of prokaryotic cells is short, the preparation cost is low, the immunogenicity to human is weak, the molecular weight is small, the penetrating power is strong, even the blood brain barrier can be penetrated, the specificity is strong, and the affinity is high. However, the preparation of the anti-rotavirus nano antibody is not reported at present.

In general, the nano antibody has great potential in the fields of detection, diagnosis and medicine, and the nano antibody targeting rotavirus obtained by screening supplements and replaces the traditional monoclonal antibody, so the nano antibody has wide application prospect in the field of biomedicine.

Disclosure of Invention

The invention aims to provide a nano antibody of a targeted rotavirus capsid protein, which has high affinity to the rotavirus capsid protein antigen, can be used for separation, detection and treatment of rotavirus, particularly research and development of a detection kit, and has good application prospect.

Generally, the amino acid sequence of the nanobody consists of about 100-130 amino acid residues, and has four framework regions (FM 1-4) and three complementarity determining regions (CDR 1-CDR 3) arranged in an alternating order. The invention discloses a nano antibody Rot1 targeting rotavirus, which comprises four framework regions and three complementarity determining regions. The complementarity determining regions of the nanobody Rot1 consist of SEQ ID NO. 1 corresponding to its CDR1, SEQ ID NO. 2 corresponding to its CDR2, and SEQ ID NO. 3 corresponding to its CDR3, respectively.

The nano antibody Rot1 targeting rotavirus protein disclosed by the invention has a decisive effect on antigen recognition by three complementarity determining regions, and four framework regions have an influence on the space structure of the complementarity determining regions so as to influence the antigen recognition. Thus, the complete amino acid sequence of the nanobody Rot1 is also an important basis for determining antigen recognition. The complete amino acid sequence of Rot1 is shown in SEQ ID NO. 4.

The nano antibody of the targeted rotavirus protein disclosed by the invention contains 124 amino acid residues, and forms a core structure of the nano antibody. The core structure of the nanobody has high affinity and specificity with the antigen protein without adding amino acid residues. In fact, the nanobody often exists in the form of a fusion protein, i.e., polypeptide and protein are added to the N-terminal or C-terminal of the core structure of the nanobody to form the fusion protein, which endows the fusion protein with more characteristics and functions while maintaining the high affinity and high specificity of the original nanobody. For example, in the panning and identification of the nanobody of the present disclosure, the C-terminal end of the nanobody is fused with a polypeptide as a protein tag (e.g., histidine tag, human influenza hemagglutinin tag), so that it can be purified using a nickel column and identified using an anti-human influenza hemagglutinin antibody. The nano antibody Rot1, a histidine tag and a human influenza hemagglutinin tag form a fusion protein, and the DNA sequence of the complete amino acid sequence is shown as SEQ ID NO. 5.

The modification of the nano antibody at the protein gene level is an obvious advantage of the nano antibody compared with the conventional antibody, and has a mature molecular biology method, and the fusion protein is also a form embodied by the common nano antibody. The polypeptide and protein which can be fused with the nano antibody are various, and comprise various protein tags (histidine tag His, human influenza virus hemagglutinin tag HA, FLAG tag, MBP tag, Myc tag and the like), green fluorescent protein, alkaline phosphatase, luciferase, glutathione transferase, toxin protein, antibody Fc fragment and the like. The fusion protein enables the nano antibody with the function of targeting antigen to have more functions, such as fusion visualization with fluorescent protein and fluorescence for tracing, fusion with cytotoxic protein to form immunotoxins (immunotoxins) for treatment and the like.

Furthermore, the nano antibody or the fusion protein constructed by the nano antibody is chemically modified, so that more characteristics and functions can be endowed to the modified nano antibody protein. For example, as described in the examples, the nanobody disclosed in the present invention is biotinylated during the identification process, so that it can be bound to avidin or streptavidin for more quantitative determination.

The nano antibody of the targeted rotavirus protein disclosed by the invention has 124 amino acid residues, has a definite structure, and the location of a modifiable group on the surface of the antibody protein is also clear, so that the nano antibody or the fusion protein constructed by the nano antibody disclosed by the invention can be chemically modified, and the commonly used modified sites are amino modified on lysine residues, carboxyl of glutamic acid and aspartic acid, and the like. In order to facilitate modification and make modification positioning and quantification more accurate, a specific chemical modification group can be introduced at the N-terminal, C-terminal or other specific part of the nanobody, and then chemically modified, for example, cysteine is introduced to obtain a thiol group of a specific modifiable site. The protein modification reaction with amino, carboxyl and sulfhydryl groups is various, and the technology is mature. Such as reaction with Fluorescein Isothiocyanate (FITC) or activated biotin, on the amino group of the nano-antibody, so as to obtain the fluorescent or biotinylated nano-antibody. The material capable of chemically modifying the nano antibody provided by the invention is various and comprises affinity chromatography filler, quantum dots, magnetic beads, colored microspheres or fluorescent microspheres, protein (such as horseradish peroxidase), chemical small molecules and the like. The requirement for the chemically modified material is to have a reactive or activatable group such as carboxyl, amino, thiol, etc. that can be used for chemical modification.

The application scenes of the specific nano antibody are many. An example is that the nano antibody Rot1 is combined with magnetic beads for concentrating and separating rotavirus. Many commercially available magnetic beads have a modifiable group such as an amino group or a carboxyl group on the surface thereof, and since a nano antibody also has a modifiable group on the surface thereof, the nano antibody can be covalently linked to the magnetic bead by a mature protein coupling method (such as a diazo method, a glutaraldehyde method, a glutaric anhydride method, a carbodiimide method, etc.). In the solution, the nano antibody recognizes and combines with rotavirus, and the conjugate of the nano antibody and the magnetic bead can capture, enrich and separate the rotavirus by using a magnetic field. The second example is the construction of a quantitative immunoassay kit by using a nano antibody Rot 1. When preparing an enzyme-linked immunosorbent assay (ELISA) kit, coating an ELISA plate with rotavirus antigen protein, adding a solution to be detected containing rotavirus, and then adding biotinylated nano antibody protein. The antigen on the rotavirus in the solution and the rotavirus antigen coated on the enzyme label plate are combined with the biotinylated nano antibody protein competitively. After washing, the amount of the remaining nano antibody capable of being combined with the rotavirus antigen coated on the ELISA plate reflects the content of the rotavirus in the solution to be detected. The amount of the nanobody can be quantified by using avidin-horseradish peroxidase (HRP) conjugated protein.

The nano antibodies disclosed by the invention are subjected to protein fusion and chemical modification, and have additional functions such as color, fluorescence, magnetism, biological activity and the like on the basis of specific identification of the rotavirus, so that the nano antibodies have wide application prospects in aspects of virus separation and concentration, virus detection, virus removal and the like.

Drawings

Various additional advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention.

Fig. 1 shows the protein electrophoresis result of nanobody Rot 1.

FIG. 2 shows the result of affinity analysis of nano-antibody Rot1 for rotavirus capsid protein antigen.

Fig. 3 shows the amino acid full-length sequence and CDR regions of nanobody Rot 1. The CDR1, CDR2 and CDR3 regions are underlined, respectively.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional reagent products which are commercially available, and manufacturers are not indicated.

Similar to coronaviruses, rotaviruses have spike proteins (spike proteins) on their surface, designated VP 4. The spike protein consists of two parts, VP5 and VP8, and VP8 is located at the top of the spike protein, and is an ideal antibody recognition site. Expression of rotavirus spike protein purification has been described in the literature (Yu et al, Chembiolchem, 16(15): 2176, 2015). We select the nano antibody targeting rotavirus protein by using the recombinant protein VP8 as protein antigen of rotavirus.

Example one panning of Nanobodies targeting Rotavirus proteins by phage display Nanobody random library

Affinity panning is carried out on the phage display library according to the modes of combination, washing, elution and amplification, and the VP8 protein coating of rotavirus is adopted as the antigen of the panning anti-rotavirus nano antibody. In the experiment, non-specific adsorption was reduced by alternating non-blocking and 5% mil blocking, and the coating concentration of the viral protein was 10. mu.g/mL. The specific steps of elutriation are as follows: mu.L of virus protein antigen with a concentration of 10. mu.g/mL (1. mu.g/well) was added to each well of the eight-linked-well enzyme standard strip and coated at 37 ℃ for 2 hours. The supernatant was discarded, and the enzyme strips were washed 5 times with 0.05% PBST, and 200. mu.L of 5% mil-k was added to each well and blocked for 2 hours at 37 ℃. The blocking solution was discarded, the enzyme strips were washed 5 times with 0.05% PBST, and 100. mu.L phage library suspension (8X 10 library capacity) was added to each well⁹pfu, titer 1X 10⁹cfu/mL), incubated for 1 hour with shaking at room temperature. Discarding the supernatant, washing the enzyme label strip 5 times with 0.05% PBST, adding 100. mu.L of 0.2M Gly-HCl elution buffer (pH 2.2) into each well for elution, standing at room temperature for elution for 10 minutes, collecting the eluate into a 1.5 mL centrifuge tube, adding 120. mu.L of 1M Tris-HCl buffer (pH 9.0) for neutralization, performing vortex mixing, taking 20. mu.L for storage as a sample for measuring titer, and amplifying all the rest samples for the next round of elutriation.

Example two, identification of Positive clones

Single colonies were randomly picked from titer-determining plates for phage rescue purification, followed by identification of positive clones by ELISA. The method comprises the following specific steps: mu.L of virus protein antigen with a concentration of 10. mu.g/mL (1. mu.g/well) was added to each well of the eight-well enzyme-labeled strip and coated at 37 ℃ for 2 hours. The supernatant was discarded, the PBST washed the enzyme strips 5 times, 200. mu.L of 5% mil k was added to each well and an additional strip was made as a negative control, and blocked for 2 hours at 37 ℃. PBST washing enzyme label strip 5 times, each hole adding 100L amplification of monoclonal phage suspension, room temperature shaking table oscillation incubation for 1 hours. The plates were washed 5 times with PBST, 100. mu.L of murine anti-HA primary antibody was added to each well, and incubated for 1 hour at room temperature with shaking in a shaker. The plates were washed 5 times with PBST, 100. mu.L of goat anti-mouse secondary antibody was added to each well, and incubated for 45 minutes with shaking in a shaker at room temperature. PBST washing 5 times, beating the liquid in the enzyme label strip as dry as possible on paper, adding 100 per wellmu.L of TMB color developing solution, and developing to a color with a proper depth. The reaction was stopped by adding 100. mu.L of 1M HCl solution to each well, and the OD was immediately read with a microplate reader₄₅₀Numerical values. Sequencing the positive sample to obtain a plurality of repeated sequences, namely a nano antibody Rot1, which is specifically shown as SEQ ID NO: 4, respectively.

Example III fusion of Nanobody Rot1 with histidine tag and human influenza Virus hemagglutinin tag

In the phage display library, the nanobody is fused with capsid protein P3 of M13 phage and displayed on the surface of phage particle. The base sequence of the obtained nano antibody Rot1 is cut into gene fragments by using restriction enzymes NdeI and XhoI, the gene fragments are inserted into pET23a plasmid, and a histidine tag and a human influenza virus hemagglutinin tag are introduced into the C terminal of the nano antibody, so that the recombinant protein can be conveniently separated, purified and identified by using a nickel column. The C-terminal amino acid sequence of the nanobody Rot1 fusion protein is: GQAGQHHHHHHGAYPYDVPDYALEHHHHHH.

EXAMPLE four expression and purification of Nanobody Rot1

The nucleotide sequence was inserted into pET23a plasmid to construct an expression vector. mu.L of the constructed pET23a plasmid containing the target gene is added into 50. mu.L of competent cells of Escherichia coli BL21 (DE 3), placed on ice for 30 minutes, then placed in a 42 ℃ water bath to be thermally shocked for 60 seconds, placed on ice for 5 minutes, and then added into the bacterial liquid with 450. mu.L of SOC culture solution. After mixing, placing the mixture in a shaking table at 37 ℃ and 200 rpm, and shaking the mixture for culture and recovery for 1 hour. Then 200 mul of the bacterial liquid is absorbed and coated on an LB + Amp solid plate, the liquid is dried, and the plate is placed upside down and cultured in an incubator at 37 ℃ overnight. The next day, single colonies were picked from overnight-cultured plates, placed in 5 mL LB + Amp medium, shake-cultured for 8 hours at 37 ℃ with 200 rpm shaking, and then the whole culture was transferred to 330 mL TB + phosphate + Amp medium, and cultured overnight at 30 ℃ with 200 rpm shaking. On the third day, the cells were collected by centrifugation, resuspended in 1 XPhosphate buffer (20 mM, pH 7.4), and then sonicated at 150 w/75%, i.e., 112.5 w, for 5 seconds with 20 seconds pauses. Then centrifuging at 20000 rpm for 20 minutes at 4 ℃, collecting ultrasonic supernatant and ultrasonic precipitation respectively, and preserving the ultrasonic supernatant at-20 ℃. The method is characterized in that the protein with higher concentration and purity is obtained by performing denaturation and renaturation on ultrasonic precipitation, and comprises the following specific steps: resuspend the sonicated samples with 10 mL of 2M urea solution and add 2M urea solution to 40 mL to wash the inclusion bodies. Centrifuge at 8000 rpm for 10 minutes at 4 ℃. The washed pellet was resuspended in 10 mL of 8M urea solution, and the inclusion bodies were solubilized by adding 8M urea solution to 35 mL, and then shaken for 1 hour at 80 rpm on a shaker at room temperature. They were then centrifuged at 10,000 rpm for 20 minutes at 4 ℃ and the supernatant (containing denatured protein of interest) was collected and the pellet was retained. Renaturation is carried out by dialysis after protein denaturation, dialysis is carried out by reducing the urea concentration in a stepped mode, 15 mL of 8M urea denaturation supernatant (target protein sample) is taken and added into 15 mL of 1 XPBS rapidly, and the concentration of the 8M urea solution is reduced to 4M. Dialysis was performed with 600 mL of 2M urea solution (stirring the dialysate in a refrigerator at 4 ℃) for 1 hour. 200 mL of the dialysate was decanted, 200 mL of 1M urea solution was added, and then dialyzed overnight. The next day, 200 mL of the dialysate was decanted, 200 mL of PBS was added, and dialysis was continued for 1 hour. 200 mL of the dialysate was decanted, 200 mL of PBS was added, and dialysis was continued for 1 hour. 200 mL of the dialysate was decanted, 200 mL of PBS was added, dialysis was continued for 1 hour, the sample was collected after dialysis, and the renatured protein solution was centrifuged at 10,000 rpm at 4 ℃ for 25 minutes. And collecting the supernatant, wherein the collected supernatant is the renatured protein solution. The result of the denatured and renatured nano antibody after protein electrophoresis is shown in figure 1, the result shows that the relative molecular mass of the nano antibody targeting rotavirus protein is about 15 kDa and is consistent with the theoretical molecular mass, and the purified band has no obvious impurity band and higher purity and meets the identification requirement.

EXAMPLE V biotinylation modification of Nanobody Rot1

NHS-Biotin reagent was weighed accurately, dissolved in 10 mM DMSO, and a sample of purified Rot1 protein was taken out, diluted to a concentration of 1 mg/mL with 1 XPBS (20 mM, pH 7.4), added in a molar ratio of 8:1 (Rot 1 protein size about 15 kDa, reduced reagent volume of 3.6. mu.L), and incubated on a shaker at room temperature and 50 rpm for 1 hour with shaking. The sample is desalted and purified after biotinylation, a Sephadex G-25 Resin column is used for purification, 1 XPBS (20 mM, pH 7.4) is used for balancing the purification column during purification, a biotinylation protein sample is added after balancing, Coomassie brilliant blue R-250 is used for detecting effluent liquid, when the effluent liquid meets the condition that Coomassie brilliant blue changes into blue, the effluent liquid is immediately collected, the PBS is used for washing the purification column after collection, and finally 20% ethanol is added for storing the purification column. And purifying and collecting to obtain the prepared biotinylated nano antibody protein sample, which is named as Rot 1-biotin. The purpose of preparing the biotinylation nano antibody protein is to develop an ELISA detection kit, and the high affinity and the high specificity of biotin and avidin (or streptavidin) can be utilized. Coating the enzyme label plate with rotavirus antigen protein expressed by the inventor, wherein rotavirus in a sample to be detected and biotinylated nano antibody protein are combined with the antigen protein competitively; under the condition that a sample to be detected exists, the biotinylation nano antibody protein capable of being combined with the antigen protein is quantified by avidin (or streptavidin) -HRP, and the amount of rotavirus in the sample to be detected is indirectly determined.

Example six detection of affinity of Nanobody Rot1 for rotavirus protein antigen

Through ELISA experiments, the nano antibody Rot1 is determined to EC50 containing rotavirus protein antigen, and a curve is made. The method comprises the following specific steps: 100. mu.L of viral protein antigen at a concentration of 10. mu.g/mL was added to each well of the eight-well enzyme strips and coated for 2 hours at 37 ℃. The supernatant was discarded, the enzyme label strip was washed 5 times with 0.05% PBST, 100. mu.L of Nanobody Rot1 (containing 0.05% Tween-20) with a concentration of 10. mu.g/mL was added to each well, the sample was diluted at 16 points in 2-fold gradient, and incubated for 1 hour with shaking at room temperature. The supernatant was discarded, the enzyme label strip was washed 5 times with 0.05% PBST, 100. mu.L of a primary antibody against the murine histidine tag diluted 1:5000 was added to each well, and incubated for 1 hour at room temperature with shaking in a shaker. The supernatant was discarded, the enzyme strips were washed 5 times with 0.05% PBST, 100. mu.L of goat anti-mouse secondary antibody diluted 1:5000 was added to each well, and incubated for 1 hour with shaking at room temperature. Discarding supernatant, washing enzyme label strip with 0.05% PBST for 5 times, patting the enzyme label strip on absorbent paper as dry as possible, adding 100 μ L of TMB color developing solution into each well, and developing according to color development conditionHolding the condition, generally 5-10 minutes, after reaching the appropriate color depth, adding 100. mu.L of 1M HCl into each well to terminate the reaction, and immediately reading OD by a microplate reader after terminating the reaction₄₅₀Finally, data analysis and plotting are performed by Microsoft excel. The ELISA result is shown in FIG. 2, the EC50 value of the nano antibody Rot1 is 194.6 ng/mL, and the affinity performance is better.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Sequence listing

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caggctccgg gtcaagagcg tgaagccgta gctgttatct ttgtgggtgg catcacttat 180

tatgctgaca gcgtaaaagg ccgttttacc attagccgcg acaacgccaa gaacaccgtt 240

accctgcaaa tgaacaacct gaaaccggaa gacaccgcca tttattactg tgccgcttat 300

gttcatatgt attgtgcttc gcagacgatt acgaggtatg actactgggg tcagggcacc 360

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Claims

1. A nano antibody targeting rotavirus protein is characterized in that a Complementary Determining Region (CDR) of an amino acid sequence of the nano antibody comprises a CDR1 shown in SEQ ID NO: 1, a CDR2 shown in SEQ ID NO: 2 and a CDR3 shown in SEQ ID NO: 3.

2. The nano antibody of the targeted rotavirus protein is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 4.

3. The nanobody of claim 1 or 2, for use in the construction of fusion proteins with polypeptides and proteins including common protein tags, green fluorescent protein, alkaline phosphatase, glutathione transferase, toxin proteins, antibody Fc fragments.

4. The nanobody of claim 1 or 2, used for the preparation of nanobody derivatives by chemical modification, comprising coupling with affinity chromatography filler, quantum dots, magnetic beads, colored or fluorescent microspheres, chemical small molecules.

5. Use of the nanobody of claim 1 or 2 in the preparation of separation and concentration medium of rotavirus, detection and diagnostic reagent, or therapeutic antibody.