CN112812190B

CN112812190B - Alpaca single-heavy-chain nano antibody resisting mouse and rabbit IgG and application

Info

Publication number: CN112812190B
Application number: CN202010417353.7A
Authority: CN
Inventors: 王玉芳; 查长春; 刘川; 刘郧飞; 王春焦; 唐静秋
Original assignee: Shanghai Baiying Biotechnology Co ltd
Current assignee: Shanghai Baiying Biotechnology Co ltd
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2022-07-08
Anticipated expiration: 2040-05-15
Also published as: CN112812190A

Abstract

The invention discloses a nano antibody for resisting mouse and rabbit IgG and application, wherein the nano antibody at least comprises one of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3, and can specifically recognize mouse and rabbit with high affinity without crossing with human IgG. Can be used for preparing the universal secondary antibody for the mice and the rabbits and has important application value.

Description

Alpaca single-heavy-chain nano antibody resisting mouse and rabbit IgG and application

Technical Field

The invention relates to a nano antibody for resisting mouse and rabbit IgG and application thereof, belonging to the technical field of biology.

Background

The antibody of the first origin, such as a mouse or a rabbit, is called a primary antibody, and the secondary antibody is an antibody capable of binding to the primary antibody, i.e., an antibody, and serves mainly to detect the presence of the antibody and amplify the signal of the primary antibody. The secondary antibody plays a great role in indirect enzyme-linked immunosorbent assay, immunochromatography and other experiments. The traditional preparation of the secondary antibody utilizes the property that the antibody is macromolecular protein with antigenicity to immunize a heterogeneous animal, and the immune globulin aiming at the antibody generated by the immune system of the heterogeneous animal has the defects of long preparation period and complex operation. The antibody library technology is a technology for cloning all antibody variable region genes of a certain animal into a plasmid or a phage for expression, and screening clones carrying specific antibody genes by using different antigens or antibodies so as to obtain corresponding specific antibodies. The antibody library technology not only can simulate the process of generating antibodies by the immune system of animals, but also has a plurality of unique advantages, the antibody library technology does not need immunization, and theoretically, 10^～10The library may contain all antibodies. The specific antibody can be directly screened from the antibody library of the non-immune animal by using the antigen or the antibody, and the antibody aiming at the self-antigen of the species can be screened.

Nanobodies, the smallest antibody molecule known to bind antigen, have a molecular weight of about 15 kD. Compared with the traditional antibody, the nano antibody has the advantages of small relative molecular mass, high affinity, high stability, good solubility, low immunogenicity, strong penetrating power, simple humanization, capability of large-scale expression in escherichia coli and the like. Therefore, the nano antibody which is simultaneously resistant to mouse and rabbit IgG and has no cross with human IgG is developed by applying the nano antibody technology, so that the nano antibody has wide application prospect.

Disclosure of Invention

In view of the above, the present invention provides a nanobody that is simultaneously resistant to mouse and rabbit IgG and does not cross human IgG, and also provides applications of the nanobody.

The invention adopts the specific technical scheme that:

a nanobody against mouse and rabbit IgG, consisting of a single heavy chain, having at least one of the following technical characteristics:

i. the heavy chain includes heavy chain CDR1, amino acid residues 26-34 of the amino acid sequence shown in SEQ ID NO. 2;

ii. The heavy chain includes heavy chain CDR2, amino acid residues 50-56 of the amino acid sequence shown in SEQ ID NO. 2;

iii, the heavy chain includes heavy chain CDR3, amino acid residues 95-105 of the amino acid sequence shown in SEQ ID NO. 2.

Preferably, the heavy chain comprises heavy chain CDR1, heavy chain CDR2, and heavy chain CDR 3; heavy chain CDR1, amino acid residues 26-34 of the amino acid sequence shown in SEQ ID NO. 2; heavy chain CDR2, amino acid residues 50-56 of the amino acid sequence shown in SEQ ID NO. 2; heavy chain CDR3 is amino acid residues 95-105 of the amino acid sequence shown in SEQ ID NO. 2.

Preferably, the amino acid sequence of the antibody is shown in SEQ ID NO. 2.

The present invention also provides:

nucleic acid encoding the nanobody against mouse and rabbit IgG as described before.

Preferably, the sequence of the nucleic acid is shown as SEQ ID NO. 1.

The present invention also provides:

expression vectors having the nucleic acids described previously, including prokaryotic expression vectors and mammalian system expression vectors.

Preferably, the prokaryotic expression vector is pET28a and the mammalian system expression vector is pcDNA3.1.

The present invention also provides:

a host cell having the expression vector described hereinbefore.

Preferably, the prokaryotic expression host cell is escherichia coli BL21, and the mammalian system expression host cell is CHO.

The present invention also provides:

the eukaryotic recombinant nanobodies described above against mouse and rabbit IgG with labels.

Preferably, the label is horseradish peroxidase (HRP).

The present invention also provides:

application of HRP-labeled anti-mouse and rabbit IgG nano-antibody in preparation of a mouse and rabbit universal secondary antibody.

The present invention also provides:

the application of the nucleic acid in preparing the nano antibody resisting mouse and rabbit IgG and the universal type secondary mouse and rabbit antibody.

The present invention also provides:

use of a host cell as hereinbefore described for the preparation of a murine rabbit universal secondary antibody.

The invention has the beneficial effects that: the invention obtains the nano antibody of the targeted mouse and rabbit IgG by the phage display technology, the antibody can specifically identify the mouse and the rabbit with high affinity, has no cross with the human IgG, can be used for preparing the universal secondary antibody of the mouse and the rabbit, and has important application value.

Drawings

FIG. 1 is a graph of the binding of enriched phage to mouse and rabbit for each round of panning in the ELISA assay of example 1.

FIG. 2 is a graph showing the binding of monoclonal phage to antigen protein in ELISA in example 1.

FIG. 3 is a graph showing the single clone enrichment ratio analysis in example 1.

FIG. 4 is a schematic diagram of prokaryotic expression vectors and mammalian system expression vectors of example 2.

FIG. 5 is the polyacrylamide gel electrophoresis detection chart of the prokaryote-expressed and mammal system-expressed nanobody of example 2.

FIG. 6 is a graph showing the results of the binding ELISA of the nano-antibody expressed in the prokaryotic expression system and the mammalian system of example 3.

FIG. 7 is a graph showing the results of detection of HRP-labeled prokaryotically expressed secondary mouse-rabbit antibody by ELISA in example 4.

Detailed Description

The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given. The methods used are conventional methods unless otherwise specified, and the reagents and materials used are commercially available products unless otherwise specified.

Example 1 screening of Nanobodies against mouse and Rabbit IgG

Respectively using Mouse IgG1, Rabbit IgG1 and Mouse IgG2a as positive screening antigens, using Human IgG as negative screening antigen, and applying phage display technology to obtain alpaca natural nano antibody phage library (the size of the library is 1.47x 10)⁹) And (4) screening the nano-antibody which resists mouse IgG and rabbit IgG and has no cross with human IgG.

Using solid phase panning, 100ug/mL Human IgG was coated onto ELISA plates at 100uL per well overnight at 4 ℃. PBST was washed three times, 200uL 3% casein was added to each well, and blocked for 2 hours at 37 ℃. The PBST three times after washing with phage display library (about 1x 10)¹²CFU), incubation for 1 hour at 37 ℃, unbound phage were collected and negative screening was repeated three times to remove phage bound to Human IgG. After being screenedThe phage were added to the blocked 100ug/mL Mouse IgG1 coated onto ELISA plates and incubated at 37 ℃ for 1 hour. Unbound phage were aspirated and washed 10 times with PBST. 100uL of glycine-hydrochloric acid (pH 2.2) was added to each well, the reaction was carried out at 37 ℃ for 7 minutes, the adsorbed phage were eluted by gently blowing the wells, and then neutralized to neutrality with Tris-HCl (pH 8.8). The eluted phage infected TG1 cells in the logarithmic growth phase, and the recovered phage were amplified for the next round of panning.

After three rounds of panning, Phage-ELISA was used to verify whether specific enrichment occurred. 2ug/mL of Mouse IgG, Rabbit IgG1, Mouse IgG2a, and Human IgG were coated onto ELISA plates and coated overnight at 4 ℃. PBST was washed three times and blocked with 3% casein at 37 ℃ for 2 hours. PBST 5 times after adding three rounds of panning phage display library, the first hole about 1x10¹²CFU, 4-fold gradient dilution, end-well blank, binding for 1 hour at 37 ℃. After 5 PBST washes, secondary HRP-labeled mouse anti-M13 antibody was added and incubated at 37 ℃ for 1 hour. And (3) washing the PBST for 5 times, adding TMB (tetramethylbenzidine) color development liquid, developing for 5-10 minutes in a dark place at room temperature, stopping developing with 2M sulfuric acid, reading the light absorption value at the wavelength of 450nm by using an enzyme-linked immunosorbent assay (ELISA), and making a Phage-ELISA binding curve.

The ELISA detection results are shown in FIG. 1, and the auxiliary phage is used as a negative control, after three rounds of enrichment, the affinity of the phage group to mouse and rabbit IgG is increased round by round, and the affinity to Human IgG is decreased round by round.

And (3) performing antigen binding analysis on the third round of enriched phage monoclonal, wherein the specific process is as follows:

TG1 cells were infected with the third round of enriched phage pool and 440 single clones were randomly picked from them, amplified and recovered. 2ug/mL of Mouse IgG, Rabbit IgG1, Mouse IgG2a, and Human IgG were coated onto ELISA plates overnight at 4 ℃. PBST was washed three times and blocked with 3% casein at 37 ℃ for 2 hours. 440 amplified monoclonal phages were incubated with 3% casein in PBST solution at a ratio of 1:1 for 1 hour at room temperature, and the incubated phages were added to the closed microplate and incubated for 1 hour at 37 ℃. After 5 PBST washes, secondary HRP-labeled mouse anti-M13 antibody was added and incubated at 37 ℃ for 1 hour. After PBST is washed for 5 times, TMB is added for color development in a dark place at room temperature for 5-10 minutes, finally the color development is stopped by 2M sulfuric acid, the light absorption value under the wavelength of 450nm is read by an enzyme-linked immunosorbent assay, and the positive clone is obtained when the light absorption value is more than twice of that of a negative control (helper phage). The monoclonal phage were analyzed for their binding ability to Mouse IgG, RabbitIgG1, Mouse IgG2a, and Human IgG, and the results are shown in FIG. 2. 59 of the 440 monoclonal phages bound to all Mouse IgG, Rabbit IgG1, Mouse IgG2 and no Human IgG (three positive and one negative).

Sequencing analysis of these 59 positive clones yielded 8 Unique sequences, with Unique1 being the dominant enriched clone (as shown in FIG. 3).

The DNA sequence of the antibody of Unique3 is SEQ ID NO:1, amino acid sequence of SEQ ID NO: 2.

SEQ ID NO：1：gaggtgcaggtggtggagtctgggggaggcttggtgcaggcgggggggtctctgagactctcctgtgtagcctctggtacgtatcgtatccatcccatggcctggtaccgccaggctccagggaaggagcgcgagttggtcgcagttattactactgatggtcagacaaggtataccgactccgtgaagggccgattcaccatttccagagccaacgacaagaacacggcggaattgcaaatgaacagcctaaagtctgaggacacaggcgtttattactgtagtctccgtaacttgaggattggatttaattactggggccaggggacccaggtcaccgtctcctcc

SEQ ID NO：2：EVQVVESGGGLVQAGGSLRLSCVASGTYRIHPMAWYRQAPGKERELVAVITTDGQTRYTDSVKGRFTISRANDKNTAELQMNSLKSEDTGVYYCSLRNLRIGFNYWGQGTQVTVSS

in the amino acid sequence, amino acid residues 26-34 (i.e., GTYRIHPMA) are the heavy chain CDR1, amino acid residues 50-56 (i.e., ITTDGQT) are the heavy chain CDR2, and amino acid residues 95-105 (i.e., SLRNLRIGFNY) are the heavy chain CDR 3.

Example 2: prokaryotic expression purification and lactation system expression purification of anti-mouse and rabbit IgG nano antibody

Prokaryotic expression purification

A prokaryotic expression vector pET28a (GST tag) of the Unique3 nanobody of example 1 was constructed as shown in FIG. 4, and then a plasmid was prepared therefrom. Plasmids were extracted and heat-shocked to strain BL21 competent cells to induce expression of nanobody proteins with IPTG. The next day, the bacterial solution was collected by centrifugation, and the cells were resuspended in 80mL of PBS and then sonicated under conditions of 200w for 3s, and then disrupted for 3s at intervals. Then, the supernatant was collected by centrifugation at 8000g at 4 ℃ and passed through GST 4FF medium (supplied inside Baiying), the nanobody was adsorbed onto the chromatography medium, 1XPBS was used to wash off the foreign proteins, 20mM reduced glutathione was added to elute, PBS was added to the obtained nanobody solution to carry out ultrafiltration (3600rpm/min, 12min, 3 times repetition), the obtained nanobody was dissolved in PBS, and SDS-PAGE was carried out on the purified nanobody, and the experimental results are shown in FIG. 5.

Mammalian system expression purification

A mammalian system expression vector pcDNA3.1 for the Unique1 nanobody of example 1 was constructed as shown in FIG. 4, and then a plasmid was prepared therefrom. The 293F cell is transfected by the successfully constructed recombinant vector by a liposome transfection method. 293F cells in logarithmic growth phase were inoculated into 6-well plates at a cell density of 1.5X 10⁶cell/mL, 37 ℃, 5% CO2 incubator plate oscillator 600rpm culture, 2 hours later for transfection. Adding the liposome-carrier mixed solution into the cell pores, culturing for 2, 4 and 6 days, supplementing materials and supplementing liquid, and collecting and purifying on day 7. The column was equilibrated with 20mL of 1xPBS at a flow rate of 1mL/min, the column was loaded at a flow rate of 1mL/min, 20mL of 1xPBS was eluted at a flow rate of 1mL/min, and the column was eluted with a citric acid buffer (pH3.4) at a flow rate of 1mL/min, collected in separate tubes, and collected at about 500uL per tube. The 10 tubes were collected together and the absorbance values at 280nm were read using a NanoDrop instrument. The high concentration protein is sucked into a dialysis bag and put into a beaker of 1XPBS for dialysis. The purified antibody was collected and the results of SDS-PAGE under reducing conditions were shown in FIG. 5.

Example 3 binding of prokaryotically and mammalian System expressed Nanobodies ELSIA

And (3) verifying the nano-antibody expressed by the prokaryotic expression system and the mammalian system by ELISA. 2ug/mL of Mouse IgG1, Rabbit IgG1, Mouse IgG2a and Human IgG were coated on ELISA plates, and were sealed at 37 ℃ for 1 hour at 4 ℃ overnight and 3% casein for 1 hour, the two systems expressing Anti-Mouse and Rabbit IgG in example 2 were diluted to 2.5ug/mL as the primary well concentration, diluted with 4-fold gradient, the final well was blank, incubated at 37 ℃ for 1 hour, PBST was washed 5 times and then dried, HRP-labeled Anti-VHH was used as the secondary antibody, incubated at 37 ℃ for 1 hour, and PBST was washed 5 times and then dried. Adding 100uL TMB into each well, reacting for 5-10min at room temperature in dark, stopping color development with 2M sulfuric acid, and reading the light absorption value at 450nm wavelength with a microplate reader.

The results are shown in fig. 6, and the nanobodies expressed by the two systems have binding to Mouse IgG1, rabbitt IgG1 and Mouse IgG2a, and have no binding to Human IgG.

Example 4 preparation of HRP-labeled murine Rabbit Universal Secondary antibody Using prokaryotically expressed anti-mouse and Rabbit IgG Nanobodies

And (3) marking the prokaryotic expression anti-mouse and rabbit IgG nano antibody according to the operation instruction of the antibody-HRP marking kit, specifically, taking the kit out of a refrigerator 30 minutes before the experiment, and balancing to room temperature (18-25 ℃). 1500mL each of CB (50mM carbonate buffer, pH 9.6, 25 ℃) and PBS (10mM phosphate, 0.9% NaCl buffer, pH 7.2, 25 ℃) were prepared. 5mg/mL pronucleus expressed anti-mouse and rabbit IgG nano antibody solution is dialyzed overnight at 4 ℃ in 50mM CB (pH 9.6), and the solution is changed for 2-3 times. 0.4mL of ultrapure water was taken out of the dark and put into an HRP tube, followed by mixing thoroughly, and 1mL of ultrapure water was taken out of the HRP tube and put into a sodium periodate (NaIO3) tube, followed by mixing thoroughly. And adding 45uL of the dissolved NaIO3 solution into the dissolved HRP solution while uniformly mixing, and standing at room temperature in the dark for reaction for 20 min. Adding 40uL of ethylene glycol into the solution, mixing uniformly, and standing at room temperature in the dark for 30 min. And adding the oxidized HRP solution into a prokaryotic expression anti-mouse and rabbit IgG nano antibody solution, fully and uniformly mixing, and dialyzing and crosslinking at room temperature for 2.5 hours. The cross-linked dialysate was 50mM CB (pH 9.6) buffer. 0.5mL of ultrapure water was added to a sodium borohydride (NaBH4) tube, and the mixture was inverted several times and mixed. And taking the crosslinked antibody-HRP solution out of the dialysis bag, placing the solution in a brown glass bottle, adding 80uL of NaBH4 solution, placing the solution at 4 ℃ in a dark place for reaction for 2 hours, and gently shaking the solution once every 30 minutes. The reduced antibody-HRP solution was dialyzed overnight (18 hours or more) at 4 ℃ against a 10mM PBS (pH 7.2). And (3) changing the liquid for 3-4 times, wherein the interval of the first liquid changing is 2 hours. The labeled antibody-HRP solution is harvested and ready for use (equal amounts of glycerol or other protein protecting agent may be added).

The ELISA plates were coated with Mouse IgG, Mouse IgG2a, Rabbit IgG and Human-IgG, respectively, 2.5ug/mL in the first well, four-fold gradient dilution, blank in the last well, and overnight at 4 ℃. Washing with PBST for 3-5 times the next day, beating to dry, adding 200uL 3% casein to each well, blocking at 37 deg.C for 1h, washing with PBST for 3-5 times, beating to dry, adding 100uL HRP-labeled nano antibody (1mg/mL, 1:2000 for dilution), incubating at 37 deg.C for 1h, washing with PBST for 3-5 times, beating to dry, adding 100uL TMB to each well, reacting at 37 deg.C in dark for 5-10min, adding 50uL 2M sulfuric acid to each well to terminate color development, and reading the absorbance at 450 nm.

The results are shown in fig. 7, the HRP-labeled pronucleus-expressed anti-Mouse and Rabbit IgG nanobodies are combined with Mouse IgG1, rabbitigg1 and Mouse IgG2a, and are not combined with Human IgG, so that the HRP-labeled pronucleus-expressed anti-Mouse and Rabbit IgG nanobodies can be used for preparing a Mouse-Rabbit universal secondary antibody.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.

Sequence listing

<110> Baiying Biotechnology Co., Ltd, Taizhou

<120> alpaca single-heavy-chain nano antibody for resisting mouse and rabbit IgG and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 348

<212> DNA

<213> Artificial sequence (Unknown)

<400> 1

gaggtgcagg tggtggagtc tgggggaggc ttggtgcagg cgggggggtc tctgagactc 60

tcctgtgtag cctctggtac gtatcgtatc catcccatgg cctggtaccg ccaggctcca 120

gggaaggagc gcgagttggt cgcagttatt actactgatg gtcagacaag gtataccgac 180

tccgtgaagg gccgattcac catttccaga gccaacgaca agaacacggc ggaattgcaa 240

atgaacagcc taaagtctga ggacacaggc gtttattact gtagtctccg taacttgagg 300

attggattta attactgggg ccaggggacc caggtcaccg tctcctcc 348

<210> 2

<211> 116

<212> PRT

<213> Artificial sequence (Unknown)

<400> 2

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Thr Tyr Arg Ile His Pro

20 25 30

Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ala

35 40 45

Val Ile Thr Thr Asp Gly Gln Thr Arg Tyr Thr Asp Ser Val Lys Gly

50 55 60

Arg Phe Thr Ile Ser Arg Ala Asn Asp Lys Asn Thr Ala Glu Leu Gln

65 70 75 80

Met Asn Ser Leu Lys Ser Glu Asp Thr Gly Val Tyr Tyr Cys Ser Leu

85 90 95

Arg Asn Leu Arg Ile Gly Phe Asn Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

Claims

1. An alpaca single heavy chain nanobody against mouse and rabbit IgG, characterized in that the nanobody heavy chain comprises heavy chain CDR1, heavy chain CDR2 and heavy chain CDR 3; heavy chain CDR1, amino acid residues 26-34 of the amino acid sequence shown in SEQ ID NO. 2; heavy chain CDR2, amino acid residues 50-56 of the amino acid sequence shown in SEQ ID NO. 2; heavy chain CDR3 is amino acid residues 95-105 of the amino acid sequence shown in SEQ ID NO. 2.

2. The alpaca single-heavy-chain nanobody against mouse and rabbit IgG according to claim 1, characterized in that the amino acid sequence of the nanobody is shown in SEQ ID NO 2.

3. Nucleic acid encoding the alpaca single-heavy-chain nanobody against mouse and rabbit IgG according to claim 1 or 2.

4. The nucleic acid of claim 3, wherein the sequence of the nucleic acid is set forth in SEQ ID NO. 1.

5. An expression vector having the nucleic acid of claim 4.

6. The expression vector of claim 5, wherein the expression vector is a prokaryotic expression vector or a mammalian system expression vector.

7. The expression vector of claim 5, wherein the prokaryotic expression vector is pET28a and the mammalian system expression vector is pcDNA3.1.

8. A host cell having the expression vector of claim 6.

9. The host cell of claim 8, wherein the host cell is e.coli BL21 or CHO.

10. The alpaca single heavy chain nanobody against mouse and rabbit IgG according to claim 1 or 2, characterized in that it has a label which is horseradish peroxidase (HRP).

11. Use of the HRP-labeled alpaca single heavy chain nanobody against mouse and rabbit IgG of claim 10 for the preparation of a murine rabbit universal secondary antibody.