CN117051002A

CN117051002A - Nanometer antibody for resisting SpyCacther003 protein and application thereof

Info

Publication number: CN117051002A
Application number: CN202310972059.6A
Authority: CN
Inventors: 蒋波; 刘彬; 叶晓华; 孙仁
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-11-14
Anticipated expiration: 2043-08-03
Also published as: CN117051002B

Abstract

The invention relates to the field of genetic engineering, in particular to an anti-SpyCacther 003 protein nanobody and application thereof, wherein the nanobody comprises at least one of SC-N2, SC-N3 or SC-N4, the nucleotide sequence of the SC-N2 is shown as SEQ ID NO.1, the nucleotide sequence of the SC-N3 is shown as SEQ ID NO.2, and the nucleotide sequence of the SC-N4 is shown as SEQ ID NO. 3. The application of the nano antibody in preparation and detection of SpyCacther003 protein products. The invention discloses a high-affinity nano antibody aiming at SpyCatcher003 protein, which has the advantages of small molecular weight, high stability, low production cost and the like, is used for detecting the existence and the location of the SpyCatcher with high sensitivity and specificity, and can be applied to the fields of laboratory research, biosensor development, clinical diagnosis and the like.

Description

Nanometer antibody for resisting SpyCacther003 protein and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to an anti-SpyCacther 003 protein nanobody and application thereof.

Background

SpyCatcher is an important tool for protein engineering and labelling, and is derived from the second immunoglobulin-like collagen adhesin domain (CnaB 2) of fibronectin (Fbab) of Streptococcus pyogenes, and is capable of specifically recognizing homologous SpyTag polypeptides and binding to form covalent links. The SpyTag/SpyCatcher system has many advantages: 1) And (3) fast combination: spyTag and Spycatcher can be rapidly combined in a few minutes at room temperature, and other auxiliary molecules are not needed; 2) The binding force is strong: covalent bonding of SpyTag and SpyCatcher forms a very firm isopeptide bond which is not easy to dissociate; 3) Reversibility: although the binding force is strong, the binding of SpyTag and SpyCatcher can be broken and re-bound by appropriate conditions. Because the SpyTag/SpyCatcher system has a plurality of advantages of simplicity, flexibility, controllability and the like, the system is widely applied to the fields of biomedical research and biotechnology at present.

However, antibodies against SpyCatcher are currently lacking in the marketplace, and only Bio-Rad (united states) provides a human-rabbit chimeric IgG subtype antibody. Generally, such conventional antibodies require high time and cost to obtain, and their large molecular weight and complex structure also limit their use in certain applications. Currently, the detection of Spycatchers and fusion proteins thereof depends more on other indirect means, which also introduces uncertainty for downstream experiments and limits the application range and convenience of the system.

In summary, although SpyCatcher has wide application in the fields of protein engineering, nanomaterial assembly, drug delivery, etc., antibodies to SpyCatcher are currently very deficient and very essential.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an anti-SpyCacther 003 protein nanobody and application thereof.

The aim of the invention is achieved by the following technical scheme: the nanometer antibody for resisting SpyCacther003 protein comprises at least one of SC-N2, SC-N3 or SC-N4, wherein the nucleotide sequence of the SC-N2 is shown as SEQ ID NO.1, the nucleotide sequence of the SC-N3 is shown as SEQ ID NO.2, and the nucleotide sequence of the SC-N4 is shown as SEQ ID NO. 3.

Further, the amino acid sequence of the SC-N2 is shown as SEQ ID NO.4, the amino acid sequence of the SC-N3 is shown as SEQ ID NO.5, and the amino acid sequence of the SC-N4 is shown as SEQ ID NO. 6.

Further, the nanobody comprises a complementarity determining region consisting of CDR1, CDR2 and CDR3.

Further, the amino acids of CDR1, CDR2 and CDR3 of the nanobody SC-N2 are respectively 26-33, 51-58 and 97-108 from the N end of SEQ ID NO. 4; the amino acids of CDR1, CDR2 and CDR3 of the nano antibody SC-N3 are the 26 th to 33 th positions, 51 th to 57 th positions and 96 th to 107 th positions of SEQ ID NO.5 from the N end respectively; the amino acids of CDR1, CDR2 and CDR3 of the nano antibody SC-N4 are the 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 108 th positions of the SEQ ID NO.6 from the N end respectively.

Further, the amino acid sequence of the SpyCacther003 protein is shown in SEQ ID NO. 7.

Further, the nucleotide sequence of an upstream primer of the amplified nano antibody is shown as SEQ ID NO.8, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 9.

The invention also provides an expression vector, which contains the nano antibody.

In one embodiment, the initial vector of the expression vector is pFUSE-hIgG1-Fc2.

The invention also provides a kit for detecting the SpyCacther003 protein, which contains the nano antibody.

The invention also provides application of the nano antibody in preparation and detection of SpyCacther003 protein products.

The invention has the following advantages: the invention discloses a high-affinity nano antibody aiming at SpyCatcher003 protein, which has the advantages of small molecular weight, high stability, low production cost and the like, fills up the gap of the market aiming at the first nano antibody of the protein, provides more and better choices for downstream application, and has various application potential and advantages. The method is used for detecting the existence and the location of the SpyCatcher with high sensitivity and specificity, and can be applied to the fields of laboratory research, biosensor development or clinical diagnosis and the like.

Drawings

FIG. 1 is a graph showing the detection of the SpyCatcher003 positive clone VHH-hFc BLI.

Detailed Description

The invention will be further described with reference to the accompanying drawings and examples, to which the scope of the invention is not limited:

phage display nanobody library:

nanobody VHH is a heavy chain antibody variable region derived from camelid body and naturally lacks a light chain, and has a molecular mass of about 15kDa, which is 1/10 of the molecular mass of conventional antibody. Nanobodies are naturally occurring minimal fragments that can bind to antigen, which have advantages such as high specificity, small size, ease of labeling and engineering, which make them of broad application prospects in diagnosis and therapy.

Phage display antibody library technology is a method for displaying proteins by using phage surfaces, and phage display nanobody libraries used in the invention are obtained by fusion expression of nanobody VHH genes derived from alpaca and P3 proteins on phage surfaces and displaying the nanobody VHH genes on the surfaces of progeny phage, and are purchased from AlpVHHS (product number: alpSDAb-P).

SpyCatcher003 protein:

SpyTag003/SpyCatcher003 mutants are the most rapidly covalently bound SpyTag/SpyCatcher systems currently. SpyCacher003 protein (N-terminal His tag) was purchased from Bio-Rad corporation (cat# TZC 025).

The SpyCacher003 protein has a sequence shown in SEQ ID NO.7, and specifically comprises the following components:

VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWIS DGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT

example 1: screening of anti-SpyCatcher 003 protein nanobody

The solid phase screening method is used for screening specific antibodies, and the specific steps are as follows:

1) Antigen plating: a4-well Maxsorph 96-well plate was coated with SpyCatcher003 protein (5. Mu.g/ml, diluted in PBS) at the appropriate concentration and overnight at 4 ℃.

2) Culturing a host bacterium: inoculating host strain TG1 into fresh 2YT culture medium with proper volume, shaking at 37 deg.C and 250rpm for 3-4 hr to OD600 = 0.5, and standing at room temperature for use.

3) The wells were subtracted from the liquid and washed 3 times with PBS.

4) The wells and phage display nanobody library AlpVHHS (Input) were blocked with MPBS (5% final concentration skim milk, PBS dilution) and incubated at room temperature for 1-2 h.

5) The blocking solution was subtracted and wells were washed 3 times with PBS.

6) Antibody libraries were added and incubated with shaking (low speed) at room temperature for binding for 2h.

7) Wells were washed multiple times with PBST and PBS, respectively.

8) Eluting: freshly prepared 100mM TEA was added and incubated for 20min at room temperature with shaking (low speed).

9) And (3) neutralization: the eluate was added to a shake tube containing 1/2 times the volume of 1M Tris-HCl (pH 7.5) and incubated at room temperature for 5min to neutralize TEA by vigorous shaking for 10 times.

10 The mixture of the previous step was added to 10 volumes of host bacteria and cultured at 37℃with shaking at 250rpm for 1 hour.

11 Phage titration: a small amount of the culture of the previous step was taken, 10-fold ratio gradient diluted with 2YT, and samples of the appropriate dilution were titrated on a 2YTA plate and incubated overnight at 37 ℃.

12 Transferring the remaining culture to a 50ml centrifuge tube, centrifuging 2500g for 10min, discarding the supernatant, adding the original volume of 2YTA resuspension bacteria solution, 37 ℃,250rpm, and continuing to culture for 2h.

13 M13KO7 helper phage was added at a final concentration of 10 ¹⁰ Phage/ml,37 ℃,250rpm infection for 30min.

14 Transfer the culture to a 50ml centrifuge tube, centrifuge 2500g for 10min, discard supernatant, add original volume of 2YTAK/IPTG (final IPTG concentration 0.2 mM) medium, 30 ℃,280rpm, incubate overnight.

15 The next day, count 11) the number of infectious monoclonal bacteria titrated, calculate the number of phages bound in this round (Output)

16 At the same time, phage in the overnight culture supernatant was collected as the second round of inputphage for the next round of screening.

17 The enrichment constant r=output/Input for each round is calculated.

When the R value is obviously increased, screening enrichment can be judged, the screening shows obvious enrichment in the 3 rd round, and the experimental result is shown in table 1.

TABLE 1SpyCatcher003 protein 3 round screening enrichment constant

Enrichment constant	First wheel R1	Second wheel R2	Third wheel R3
				Output/Input	1.64×10 ^-7	1.25×10 ^-7	5.89×10 ^-3

Example 2: positive monoclonal identification of anti-SpyCatcher 003 protein nano antibody-PhageELISA

1) The output phase of the previous round of the significant enrichment round was selected to infect TG1 bacteria, and example 1 shows that this time significant enrichment occurred in round 3, so the output phase of the second round was used.

2) TG1 host bacteria were infected with an appropriate amount of Outputphage, plated on LB/Amp100 dishes, and cultured overnight at 37 ℃.

3) The next day, several (e.g., 192) single clones were picked from LB/Amp100 plates and added to 96-well deep well plates containing 200. Mu.L/well 2YT/Amp100/2% glucose medium, 250rpm,37℃and incubated overnight.

4) The overnight cultures from the previous day were inoculated at a 1:50 ratio into 96-well deep well plates containing 200L/well 2YT/Amp100/2% glucose medium at 250rpm at 37℃until the inoculum OD600 ≡0.5 (the cells were in logarithmic growth phase).

5) Adding a final concentration of about 10 ¹⁰ Phage/ml M13K07 helper phage, 37 ℃,250rpm, 30min infection.

6) 3000g, RT, centrifuge for 10min, reverse-buckling to discard supernatant, add 400L/well 2YT/Amp100/Kan50, re-suspend the thallus.

7) Culturing at 37℃at 250rpm overnight. Coating 96-well plates for ELISA in open sun with 2 μg/ml SpyCatcher003 protein, 100L/well; coating overnight at 4 ℃.

8) The yesterday coated 96-well ELISA plate was removed, antigen was subtracted, and washed 3 times with PBS, 200L/well.

9) The ELISA plate was blocked by addition of MPBS at 200L/well, room temperature for 2h.

10 Taking out the 96-well deep well plate in the step 3), and centrifuging for 10min at 3000 g.

11 60L/well supernatant was aspirated into a clean 96-well plate, and an equal volume of 5% skim milk-PBS solution was added, pre-blocked phase-containing culture supernatant, RT, for 1-2 h.

12 Blocking solution in ELISA plates was discarded, washed 4 times with PBST, 200L/well.

13 Add pre-blocked culture supernatant from 14, 100L/well, incubate with gentle shaking at room temperature for 1h.

14 8 washes with PBST, 200L/well.

15 HRP-anti-M13 antibody, 100L/well, and incubated for 30min with gentle shaking at room temperature.

16 6 washes with PBST and 2 washes with PBS, 200L/well.

17 TMB substrate, 50L/well, and incubating at room temperature (25deg.C) or 37deg.C in the dark for 5-10 min, and observing color development (blue).

18 An equal volume of stop solution (1M sulfuric acid solution), 100L/well, was added to stop the reaction, and the reaction solution in the well was changed from blue to yellow.

19 30min after termination of the reaction, absorbance at 450nm was measured with a microplate reader.

20 Positive clones (OD 450 value. Gtoreq.0.5) were picked and sequenced.

21 Comparing the monoclonal sequences to obtain positive monoclonal (Unique) with different CDR3 sequences, and obtaining 3 specific monoclonal (named SC-N2-SC-N4) in total in the screening.

The SC-N2 nucleotide sequence is shown as SEQ ID NO.1, and concretely comprises:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCATT GAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGGATCTATGCCGTGAATTGGGTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTCTAGCACGTATTACCTGGACTACTGGTAGTGTATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAAGGACAACTCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAATATGAGGACACGGCCGTTTATTACTGCGCATTCCGCTCCGTGGGCGACTCCAAATATTCCGGCTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA-3’

the SC-N3 nucleotide sequence is shown as SEQ ID NO.2, and concretely comprises:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCATT GAGACTCTCCTGTGCAGCCTCTGGAAGCATCTTCAGGATCTATGCCGTGAATTGGTCCCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCCCGTATTAGTCGTAGTGGCAGCACATACTACGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAAAGACAATACCGAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCCCGGGGTGCTGGTTCCCCCCATTATGACTATTGGGGCCAGGGGACCCAGGTCACTGTCTCCTCA-3’

the SC-N4 nucleotide sequence is shown as SEQ ID NO.3, and concretely comprises:

5’-CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCATGGGGGGTCCCT GAGACTCTCCTGTGCAGCCTCTGGAAGCATCAGCAGTATCTATGCCATGGGCTGGGTCCGTCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGGAGCTACTAGCTGGAGTGGTGGTAGTACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTTCAGCTAGACCGGTCTCGACCCCAAGGTGGGACTACTGGGGCCAGGGGACCCAGGTCA CTGTCTCCTCA-3’

the SC-N2 amino acid sequence is shown as SEQ ID NO.4, and concretely comprises:

the SC-N3 amino acid sequence is shown as SEQ ID NO.5, and concretely comprises:

the SC-N4 amino acid sequence is shown as SEQ ID NO.6, and concretely comprises:

in the above amino acid sequence, the bolded part is CDR1, the single underlined bolded part is CDR2, and the double underlined bolded part is CDR3.

Example 3: verification of anti-SpyCatcher 003 protein nanobody binding activity

To further confirm the binding activity of the positive monoclonal, eukaryotic expression was performed on these 3 positive clones and their binding to SpyCatcher003 protein was further verified.

1. Eukaryotic expression

Eukaryotic expression is carried out in a form of nanometer antibody (VHH) fused with human IgG1 subtype antibody finger-Fc (hFc) fragment, and expression products are VHH-hFc dimer, and the specific expression steps are as follows:

1) Recombinant vector construction:

eukaryotic expression vector pFUSE-hIgG1-Fc2 (purchased from InvivoGen under the trade designation pFUSE-hg1Fc 2)) was double digested with EcoRV and NcoI;

positive VHH clone fragments were amplified by PCR, with specific reaction conditions as shown in table 2:

TABLE 2PCR reaction System and reaction conditions

* The primer sequences were as follows:

VHH-Fc F：

5’-GCACTTGTCACGAATTCGATACTCAGGTGCAGCTCGTGGAG-3’SEQ ID NO.8

VHH-Fc R：

5’-TGAGTTTTGTCAGATCTAACTGAGGAGACRGTGACCTGG-3’SEQ ID NO.9

the positive VHH fragment is connected to an enzyme tangential vector through homologous recombination after being purified by a PCR product recovery kit, the connection product is transformed into DH5 alpha competent cells, 500 mu LSOC heavy suspension cells are added, the temperature is 37 ℃, the rpm is 220, after 30 minutes of activation, 100 mu L of bacterial liquid is taken to coat an LB agarose plate containing bleomycin (Zeocin) with the final concentration of 50 mu g/mL, and the mixture is incubated overnight at 37 ℃; the next day, 4 clones were randomly picked each and sequenced by the company engine.

2) Transfection: recombinant expression vectors were transfected into 293F cells using PEI.

3) Culturing: the cells were cultured for 5 days.

4) Antibody separation: culture supernatants were collected and VHH-hFc isolated and purified using ProteinA.

5) The antibodies were solution-displaced and concentrated using ultrafiltration tubes.

6) Measuring concentration, packaging, and storing in ultralow temperature refrigerator.

Example 5: determination of nanobody affinity by enzyme-linked immunosorbent assay (ELISA)

The affinity of VHH-hFc to SpyCacther003 protein was determined using ELISA as follows:

1) Coating: spyCacther003 protein was diluted to 2. Mu.g/mL, 100. Mu.L/well using PBS and bound overnight at 4 ℃.

2) Washing: the PBS was washed 3 times, 200. Mu.L/well.

3) Closing: 5% MPBS, 200. Mu.L/well, incubated at room temperature for 2h.

4) Incubation resistance: the gradient diluted VHH-hFc antibody was bound for 1h at room temperature at 100. Mu.L/well.

5) Washing: wash 6 times with PBST, 200 μl/well.

6) Secondary antibody incubation: anti-human IgG-Fc antibodies labeled with horseradish peroxidase (HRP), 1: diluted at 5000, 100. Mu.L/well, combined at room temperature for 30min.

7) Washing: the cells were washed 6 times, 3 times, 200. Mu.L/well with PBST followed by PBS.

8) Color development: TMB substrate, 50. Mu.L/well, was added and reacted at room temperature for about 5min.

9) And (3) terminating: 1M dilute sulfuric acid, 50. Mu.L/well, was added.

10 Reading OD value): OD450 signal values were determined using a microplate reader.

11 Data analysis as shown in table 3.

TABLE 3SpyCatcher003 positive clone affinity (ELISA)

Numbering device	SC-N2	SC-N3	SC-N4
				EC50(μg/mL)	0.012	0.253	0.450

From the ELISA results, it can be seen that: all 3 monoclonal had very high affinity.

Example 6: determination of nanobody affinity by biological film interference technique (Bio-LayerInterferometry, BIL)

Using SidorisBLISsystems, spyCactThe affinity of VHH-hFc was determined using her003 protein (with 6X histag, 5. Mu.g/mL) as ligand (ligandd) and 2-fold gradient dilution of VHH-hFc as analyte (10. Mu.g/mL, 5. Mu.g/mL, 2.5. Mu.g/mL, 1.25. Mu.g/mL, 0.625. Mu.g/mL, 0.3125. Mu.g/mL, 0.15625. Mu.g/mL, 7 total concentrations) and the experimental results are shown in FIG. 1 and Table 4.

TABLE 4SpyCatcher003 positive clone VHH-hFc affinity (BLI)

Numbering device	SC-N2	SC-N3	SC-N4
				KD(nM)	0.45	1.37	1.41

As can be seen from table 4 and fig. 1: three clones all had very high affinity (nM level), with the highest affinity for SC-N2.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.

Claims

1. The nanometer antibody for resisting the SpyCacther003 protein is characterized by comprising at least one of SC-N2, SC-N3 or SC-N4, wherein the nucleotide sequence of the SC-N2 is shown as SEQ ID NO.1, the nucleotide sequence of the SC-N3 is shown as SEQ ID NO.2, and the nucleotide sequence of the SC-N4 is shown as SEQ ID NO. 3.

2. The nanometer antibody against SpyCacther003 protein according to claim 1, wherein the amino acid sequence of SC-N2 is shown in SEQ ID NO.4, the amino acid sequence of SC-N3 is shown in SEQ ID NO.5, and the amino acid sequence of SC-N4 is shown in SEQ ID NO. 6.

3. The nanobody against SpyCacther003 protein according to claim 1, wherein said nanobody comprises a complementarity determining region consisting of CDR1, CDR2 and CDR3.

4. The nanobody against SpyCacther003 protein according to claim 1, wherein the amino acids of CDR1, CDR2 and CDR3 of said nanobody SC-N2 are SEQ ID No.4 from positions 26-33, 51-58 and 97-108, respectively; the amino acids of CDR1, CDR2 and CDR3 of the nano antibody SC-N3 are the 26 th to 33 th positions, 51 th to 57 th positions and 96 th to 107 th positions of SEQ ID NO.5 from the N end respectively; the amino acids of CDR1, CDR2 and CDR3 of the nano antibody SC-N4 are the 26 th to 33 th positions, 51 th to 58 th positions and 97 th to 108 th positions of the SEQ ID NO.6 from the N end respectively.

5. The nanobody against SpyCacther003 protein according to claim 1, wherein the amino acid sequence of the SpyCacther003 protein is shown in SEQ ID No. 7.

6. The nanobody against SpyCacther003 protein according to claim 1, wherein the nucleotide sequence of the upstream primer for amplifying the nanobody is shown in SEQ ID No.8, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 9.

7. An expression vector comprising the nanobody of any one of claims 1-6.

8. The expression vector of claim 7, wherein the initial vector of the expression vector is pFUSE-hig 1-Fc2.

9. A kit for detecting SpyCacther003 protein, comprising the nanobody of any of claims 1-6.

10. Use of the nanobody of any of claims 1-6 for the preparation of a product for detecting SpyCacther003 protein.