CN118085077A - Nano antibody BSA-4-41 for resisting bovine serum albumin and application thereof - Google Patents

Abstract

The invention discloses a nanometer antibody BSA-4-41 of anti-bovine serum albumin (Bovine Serum Albumin, BSA) and application thereof. The nano antibody BSA-4-41 comprises protein or polypeptide with an amino acid sequence shown as SEQ ID NO.1, is obtained by screening a phage display natural nano antibody library, has higher affinity and can be combined with BSA. The nano antibody provided by the invention can be applied to directional binding, immunodetection, enrichment purification and the like of BSA; the amino acid sequence provided by the invention can be used as a precursor, and can be modified by random mutation or site-directed mutation technology to obtain mutants with better properties such as affinity, expression quantity, water solubility, specificity, stability and the like.

Description

Nano antibody BSA-4-41 for resisting bovine serum albumin and application thereof

Technical Field

The invention relates to the technical field of nanobodies, in particular to a nanobody BSA-4-41 for resisting bovine serum albumin and application thereof.

Background

In camelidae or cartilaginous fish animals, there is a heavy chain antibody (HEAVY CHAIN anti-body, HCAb) with a naturally deleted light chain, and the variable region (Variable domains of HEAVY CHAIN of HEAVY CHAIN anti-body, VHH) is a single domain heavy chain antibody, which is also called a nanobody due to its small size. Nanobodies are currently known antigen-binding fragments with the smallest molecular weight, the molecular weight of which is only 15kDa, which is about one tenth of that of conventional IgG antibodies, and have many other advantages, such as high stability, strong water solubility, small volume, resistance to organic solvents, easy modification, and recognizable cryptic epitopes, which are different from conventional antibodies.

Bovine serum albumin (Bovine Serum Albumin, BSA) is a globulin extracted from bovine serum, is the most abundant protein component in serum, consists of 583 amino acid residues, has a molecular weight of about 66.43kDa and has an isoelectric point pI of 4.7.BSA has higher stability in solution and thermal stability, and has a plurality of important applications in researches such as biology, biomedicine and the like, for example, the BSA can be used as a blocking agent in immunoassay, can also be used as a carrier protein for coupling small molecular compounds so as to prepare and obtain complete antigens, and is used for immunizing animals or constructing an immunoassay method. In addition, bovine serum is an important component of the culture broth in the current cell culture, while BSA is a major component in bovine serum, and also is one of the major factors causing allergic reaction as a heterologous protein.

Therefore, detection and removal of BSA in a cell solution cultured with bovine serum has an important role in improving the biosafety of a cell biologicals; the preparation and development of the anti-BSA antibody are important for directional fixation, purification, detection and the like of BSA.

Disclosure of Invention

The invention aims to provide a nano antibody BSA-4-41 for resisting BSA and application thereof, and overcomes the defects of complex preparation process and high cost of the existing polyclonal antibody and monoclonal antibody.

In one aspect, the invention provides an anti-BSA nanobody BSA-4-41 comprising an amino acid sequence as shown in SEQ ID NO. 1.

Optionally, the nanobody comprises at least one of a monomer, a bivalent antibody, and a multivalent antibody.

In a second aspect, the invention provides a gene encoding nanobody BSA-4-41, the nucleotide sequence of which is shown in SEQ ID NO. 9.

In a third aspect, the invention provides the use of the nanobody-encoding gene in the construction of an expression vector.

In a fourth aspect, the present invention provides a drug conjugate comprising the nanobody, wherein the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

In a fifth aspect, the present invention provides the use of the nanobody BSA-4-41 in a pharmaceutical composition.

In a sixth aspect, the invention provides the use of the nanobody BSA-4-41 in immunodetection.

In a seventh aspect, the present invention provides a recombinant protein constructed by the nanobody BSA-4-41.

The beneficial effects of the invention include:

(1) The preparation method of the nano antibody provided by the invention has the advantages of simple flow and low cost;

(2) The nano antibody provided by the invention is derived from phage display natural nano antibody library (AlpSDab-P) of Sichuan apak biotechnology limited company, and has the advantages of large library capacity, strong diversity and the like;

(3) The nano antibody provided by the invention can be specifically combined with BSA, and can be applied to the fields of BSA directional combination, immunodetection, enrichment purification and the like;

(4) The amino acid sequence of the nano antibody provided by the invention can be used as a precursor, and can be modified by random or site-directed mutagenesis technology to obtain mutants with better properties (water solubility, yield, affinity, specificity, stability and the like).

Drawings

FIG. 1 shows the results of a phase-ELISA to identify the third round of screening anti-BSA positive cloned phages in example 1;

FIG. 2 shows the results of a phase-ELISA to identify the anti-BSA positive clone phage from the fourth round of screening in example 1;

FIG. 3 shows the result of Western Blot identification of anti-BSA of nanobody BSA-4-41 of example 1;

FIG. 4 is a schematic diagram of the amino acid sequence and domain of the anti-BSA nanobody BSA-4-41 prepared in example 2;

FIG. 5 is a SDS-PAGE identification of anti-BSA nanobody BSA-4-41 prepared in example 2.

Detailed Description

The invention will be further illustrated by the following examples in conjunction with the accompanying drawings.

In a first aspect, embodiments of the present invention provide a method for screening anti-BSA nanobody BSA-4-41.

In a second aspect, embodiments of the present invention provide an anti-BSA nanobody BSA-4-41 comprising an amino acid sequence as shown in SEQ ID NO. 1.

In some embodiments, nanobody BSA-4-41 includes four Framework Regions (FRs) and three complementarity determining regions (Complementarity-DETERMINING REGION, CDRs); the amino acid sequences of the framework regions (FR 1-FR 4) are shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8; the amino acid sequences of the complementarity determining regions (CDR 1-CDR 3) are shown as SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7; the CDR region is mainly responsible for BSA recognition, the FR region structure is relatively stable, and the structure of the nanobody is mainly maintained.

In some embodiments, the nanobody comprises at least one of a monomer, a bivalent antibody, a multivalent antibody.

In some embodiments, the nucleic acid molecule of the nanobody comprises DNA, RNA, cDNA.

In some embodiments, the amino acid sequence can be used as a precursor, and modified by random or site-directed mutagenesis techniques to obtain mutants with better properties in terms of yield, water solubility, affinity, specificity, stability, etc.

In a third aspect, embodiments of the present invention provide a gene encoding nanobody BSA-4-41, the nucleotide sequence of which is shown in SEQ ID NO. 9.

In a fourth aspect, the present embodiment provides an application of the gene encoding the nanobody in constructing an expression vector.

In some embodiments, the nucleic acid sequences are 85-90% similar due to degeneracy of the genetic code.

In a fifth aspect, embodiments of the present invention provide a drug conjugate comprising the nanobody, where the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

In a sixth aspect, embodiments of the present invention provide the use of the nanobody BSA-4-41 in a pharmaceutical composition.

In a seventh aspect, embodiments of the present invention provide recombinant proteins constructed from the nanobody BSA-4-41.

In some embodiments, the recombinant protein is expressed by a host cell, including E.coli, yeast cells, mammalian cells.

Specifically, the expression of the host cell may include the integration of a nucleic acid sequence encoding a BSA-4-41 nanobody into the genome of the host cell, including the expression vector.

Specifically, the host cell is capable of expressing at least one of the nanobody and a polypeptide comprising the amino acid sequence of the nanobody.

In an eighth aspect, the embodiment of the invention provides an application of the nanobody BSA-4-41 in immunodetection.

In some embodiments, the application includes directed binding, immunodetection, and enrichment purification of BSA.

Specifically, the application of the directional binding of BSA comprises the directional binding of various conjugates, such as coating antigens, by using the nano antibody BSA-4-41 provided by the invention and taking BSA as carrier protein;

specifically, the application of the immune detection of BSA comprises the construction of a related immune detection method by using the nano antibody BSA-4-41 provided by the invention as a detection reagent.

Specifically, the enrichment and purification application of BSA comprises the step of utilizing the nanobody BSA-4-41 provided by the invention as a capturing element to realize enrichment and purification of BSA.

The culture mediums in the embodiment of the invention are all commercially available, the phage display natural nano antibody library is from Sichuan apak biotechnology Co., ltd, and the 2 XYT culture medium is from Beijing Soy treasure technology Co., ltd; the self-induction medium was from apaker biotechnology limited, sichuan.

Example 1

The embodiment 1 of the invention provides a solid phase panning method of a nano antibody, which comprises the following steps:

S1, BSA was diluted to the desired concentration by carbonate buffer at ph=8.6, the concentrations are shown in table 1; 100 μl of BSA was added to a 96-well plate and incubated at 4deg.C for 12h; after incubation was completed, 96-well plates were washed 3 times with PBST solution;

S2, after washing, adding 300 mu L of 1% gelatin sealing liquid into each hole, and incubating for 2 hours at 37 ℃; after incubation was completed, 96-well plates were washed 3 times with PBST solution;

S3, after washing, adding 100 mu L of diluted phage display natural nano antibody library (AlpSDab-P) into each hole, wherein the library input amount is 1X 10 ¹¹ pfu/hole, and incubating for 1h at 37 ℃; after incubation was completed, the 96-well plates were washed 10 times with PBST solution;

s4, after washing, adding 94.5 mu L of Gly-HCl eluent with pH=2.2 into each hole, and incubating for 8min on a horizontal shaking table at room temperature;

S5, after the culture is completed, absorbing 94.5 mu L of eluent from each hole, and adding the eluent into 5.5 mu L of Tris-HCl neutralizing buffer solution with pH=9.0 for neutralization to obtain 100 mu L of neutralizing solution;

S6, taking 10 mu L of the neutralization solution, and measuring the titer of the eluted phage on a2 XYT/Amp resistance plate; the remaining 90 μl of neutralization solution was used to infect e.coli ER2738 for phage amplification and the resulting phage was used for the next round of screening;

In order to obtain the nanobody with high affinity with antigen, four rounds of panning are carried out together, the panning conditions and experimental parameters of each round are shown in table 1, and the phage vector pComb3 XSS-anti-BSA nanobody is obtained after the fourth round of screening.

TABLE 1 panning conditions and experimental results for anti-BSA nanobodies

Example 2

The embodiment 2 of the invention provides a method for expressing anti-BSA nanobody protein BSA-4-41 in an auto-induction mode, which comprises the following steps:

D1, converting phage vector pComb3XSS-BSA-4-41 into chemically competent cells E.coli Rosetta by a heat shock method at 42 ℃ for 90s, coating a2 XYT/Amp resistance plate, and picking a single colony after 1D;

d2, adding the single colony and 5 mu L of 100mg/mL ampicillin (Amp) into 5mL of 2 XYT liquid culture medium, and carrying out shake culture for 12h at 37 ℃ with the final concentration of the Amp of 100 mu g/mL and 180rpm to obtain a first bacterial liquid after the culture is completed;

D3, inoculating the first bacterial liquid into 100mL of self-induction culture medium with an inoculum size of 1% (v/v), and adding 50 mu L of 100mg/mL of Amp, wherein the final concentration of Amp is 50 mu g/mL; shaking culture at 37deg.C and 180rpm for 3 hr to logarithmic phase (OD ₆₀₀ reaching 0.5-0.7) to obtain culture one;

D4, placing the first culture at 23 ℃ and shaking culture at 1-30 rpm for 12 hours to induce protein expression to obtain a second culture;

D5, centrifuging the second culture at 8000rpm/min for 10min; re-suspending the precipitate obtained by centrifugation through 20mL of purified balance buffer solution to obtain a bacterial body weight suspension;

d6, adding 20mg of lysozyme into the heavy suspension, wherein the final concentration of the lysozyme is 1mg/mL, and carrying out enzymolysis on the heavy suspension for 30min at 4 ℃ through a horizontal shaking table to obtain an enzymolysis solution;

D7, placing the enzymolysis liquid in a beaker filled with an ice-water mixture, crushing for 30min by using a cell ultrasonic crusher, wherein the crushing condition of the cell crusher is that the power is 200W, and working for 3 seconds and intermittent for 12 seconds; observing that the enzymolysis liquid becomes clear and transparent from turbidity, and stopping crushing to obtain a crushed product;

D8, centrifuging the crushed product at 13000 Xg and 4 ℃ for 30min, and collecting protein supernatant; filtering the supernatant with a 0.22 μm water-based filter membrane, removing impurities, and purifying;

The purification in step D8 comprises the steps of:

D81, adding 2mL of Ni-NTA column material into the purification column, washing with at least 5 times of purified water with the volume of the column after Ni-NTA naturally subsides, removing impurities, and balancing the purification column by using 15mL of balancing buffer solution;

d82, adding the supernatant to a purification column in batches, combining the protein supernatant with Ni-NTA for 5min, slowly flowing out, and collecting the flow-through liquid of each time;

D83, balancing the purification column by using a balancing buffer (containing imidazole) to achieve the purpose of eluting the protein, wherein the elution is gradually carried out from low imidazole concentration to high imidazole concentration, the low imidazole concentration is used for eluting the hybrid protein combined in the purification column, and each imidazole concentration is at least 20mL;

The first 5mL of eluting buffer solution with concentration of D84 and 250mmol/L imidazole is used for eluting the target protein combined in the purification column, and the target protein solution is collected and stored in a centrifuge tube;

D85, transferring the collected target protein solution into a 3kDa dialysis bag for desalting, and putting the target protein solution into PBS (phosphate buffered saline) dialysate with the pH value of 7.4 and the concentration of 10mmol/L at a refrigerator at the temperature of 4 ℃ for dialyzing, wherein the dialysate is replaced every 8 hours, and the dialyzing time is 1D;

D86, concentrating the protein liquid by using a 3kDa ultrafiltration tube after dialysis is completed, centrifuging for 10 minutes at 4 ℃ and 3000g/min until the final protein liquid is kept at about 1mL, and measuring the protein concentration by using a Nanodrop 2000 micro-spectrophotometer; after the measurement, glycerin was added at a final glycerin concentration of 50%, and the mixture was packaged in 500. Mu.L centrifuge tubes and frozen in a refrigerator at-80 ℃.

Property detection

1. Positive clone identification of nanobodies

(1) Positive clones were identified by phage-ELISA comprising the steps of:

In the panning of round 3 and round 4 of example 1, 48 individual clones were randomly selected on plates for phage titer determination for phage amplification, respectively;

Experimental group: the BSA concentration was diluted to 10. Mu.g/mL using PBS buffer, added to 96-well plates, 100. Mu.L of BSA per well, i.e., 1. Mu.g of BSA per well, and incubated overnight at 4 ℃;

blank group: the gelatin concentration was diluted to 10. Mu.g/mL using PBS buffer, 100. Mu.L was added to each well and incubated overnight at 4 ℃;

After coating was completed, washing 3 times with PBST solution; adding 5mLTween-20 to 1L PBS buffer solution of PBST solution, wherein the final concentration of PBS in the PBST solution is 10mmol/L;

after washing, 300 μl of 1% gelatin was added to each well and the wells were blocked at 37deg.C for 2h; after the sealing is completed, PBST is added for washing 3 times;

After washing, 100 μl phage amplification solution was added to each well of 96-well plates of the experimental and control groups, and incubated at 37deg.C for 1h; after the incubation is completed, PBST is added for washing 3 times;

After washing, 100. Mu.L of diluted anti-M13-HRP secondary antibody (final concentration 0.275. Mu.g/mL) was added to each well, and incubated at 37℃for 1h; after the incubation is completed, adding PBST solution for washing for 4 times;

After the washing is finished, 100 mu L of TMB color developing solution is added into each hole, and the color development reaction is carried out for 10min at 37 ℃ in a dark place; after the color development reaction is finished, 50 mu L of 2mol/L H ₂SO₄ stop solution is added into each hole;

Measuring an absorption value at 450nm by using an enzyme label instrument; the phage clones of the experimental group with OD ₄₅₀ 10 times greater than that of the control group were positive clones, as shown in FIG. 1 and FIG. 2, FIG. 1 shows the results of the third round of panning of 1-48 monoclonal phase-ELISA, and FIG. 2 shows the results of the fourth round of panning of 1-48 monoclonal phase-ELISA.

(2) WestemBlot identification of positive clone sequencing results:

sequencing the BSA-4-41 positive clone, and identifying the binding effect of the BSA-4-41 positive phage on BSA with different concentrations by using Western Blot, wherein the result is shown in FIG. 3;

Referring to FIG. 3, lanes M are Maker, lanes 1-3 are 1 μg,5 μg, 10 μg BSA protein, respectively; as shown by the arrow, westernBlot shows that lanes 1-3 all appear as bands at 63kDa upward and darken in color as the BSA content increases, indicating that phage BSA-4-41 is able to bind BSA;

The amino acid sequence of the nano antibody BSA-4-41 is shown as SEQ ID NO.1, and comprises a framework region (FR 1-4) and a complementarity determining region (CDR 1-3), and the number and the structural domain of the amino acid sequence IMGT are schematically shown as figure 4;

The amino acid sequence of the framework region (FR 1-FR 4) of the nano antibody BSA-4-41 is shown as SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO. 8; the amino acid sequences of the complementarity determining regions (CDR 1-CDR 3) are shown as SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7; the CDR region is mainly responsible for the recognition of BSA, the FR region structure is relatively stable, and the structure of the nano antibody is mainly maintained;

the nucleotide sequence of the nano antibody BSA-4-41 is shown as SEQ ID NO. 9.

2. Identification of the anti-BSA nanobody BSA-4-41

Identification was performed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting (WesternBlot);

SDS-PAGE results are shown in FIG. 5, wherein lane M is Maker, and lane 1 is nanobody BSA-4-41 obtained in example 2;

The results in FIG. 5 show that the BSA-4-41 nanobody was successfully expressed and purified, and the nanobody was of higher purity, while the molecular weight of the nanobody was close to that predicted by software, as indicated by the arrow in FIG. 5, at about 15kDa.

The analysis result shows that the nano antibody BSA-4-41 of the anti-BSA obtained by screening can be combined with BSA, and has stronger specificity. Meanwhile, the amino acid sequence of the nano antibody BSA-4-41 provided by the invention can be used as a precursor, and can be modified by a random or site-directed mutagenesis technology to obtain mutants with better properties in various aspects such as yield, water solubility, affinity, specificity, stability and the like.

While embodiments of the present invention have been described in detail hereinabove, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. It is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention described herein is capable of other embodiments and of being practiced or of being carried out in various ways.

Claims (8)

1. A nano antibody BSA-4-41 of anti-bovine serum albumin, which is characterized in that the nano antibody comprises an amino acid sequence shown as SEQ ID NO. 1.

2. The nanobody of claim 1, wherein the nanobody comprises at least one of a monomer, a bivalent antibody, a multivalent antibody.

3. The gene encoding the nanobody BSA-4-41 according to claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 9.

4. Use of the gene according to claim 3 for constructing an expression vector.

5. The drug conjugate of nanobody composition of claim 1, wherein the drug conjugate comprises at least one of a label, a drug, a cytokine, a nanomaterial, and a functional protein.

6. Use of nanobody BSA-4-41 according to any of claims 1-2 in a pharmaceutical composition.

7. Use of nanobody BSA-4-41 according to any of claims 1-2 in immunoassays.

8. Recombinant protein constructed by nanobody BSA-4-41 according to any of claims 1-2.