CN116217722B - Nanometer antibody of anti-cyclin E protein, encoding gene and application - Google Patents

Abstract

The invention relates to a nanometer antibody of an anti-cyclin E protein, a coding gene and application thereof, and relates to the field of genetic engineering. The amino acid sequence of the nano antibody comprises a framework region sequence and a complementary determining region sequence, wherein the framework region sequence is selected from SEQ ID NO:1-4, the complementarity determining region sequence is selected from the group consisting of SEQ ID NOs: 5-7. The anti-CyclinE nanometer antibody has a small molecular weight of about 16kDa, but has high antigen binding force and specificity.

Description

Nanometer antibody of anti-cyclin E protein, encoding gene and application

Technical Field

The invention relates to the field of genetic engineering, in particular to a nanometer antibody of an anti-cyclene protein, a coding gene and application thereof.

Background

Cyclin e proteins are a highly conserved class of cyclin proteins involved in regulating the transition from G1 phase to S phase in the cell cycle. The cyclin E protein changes periodically throughout the cell cycle, its expression is undetectable in the G0 phase, peaks at the G1/S junction, and begins to decline when entering the S phase. The expression of human cyclin E in normal proliferating untransformed cells is so low that it is undetectable, whereas the expression in proliferating cells, tumor cells and malignant transformed cells is significantly elevated, positively correlated with the malignancy of the tumor. Drosophila CyclinE protein consists of 709 amino acids and is about 71kda in size.

Drosophila melanogaster is a model organism of genetic and developmental biology, and antibodies play an important role in the basic biological research process of Drosophila melanogaster. Because drosophila protein has low homology with mammalian protein, some antibodies against proteins of human, murine or rabbit origin are not suitable for drosophila, and thus the preparation of drosophila protein antibodies is significant.

Disclosure of Invention

In view of the above problems, the present invention provides an anti-cyclin e nanobody having a small molecular weight of about 16kDa but high antigen binding capacity and specificity.

In order to achieve the above object, the present invention provides a nanobody against a cyclin e protein, the amino acid sequence of the nanobody comprises a framework region sequence and a complementarity determining region sequence, the framework region sequence is selected from the group consisting of:

MADVQLQESGGGLVQAGGSLRLSCAASG(SEQ ID NO：1)

FRQAPGKEREFV(SEQ ID NO：2)

YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC(SEQ ID NO：3)

WGQGTQVTVSS(SEQ ID NO：4)；

the complementarity determining region sequence is selected from the group consisting of:

GTFSRYSMGW(SEQ ID NO：5)

SAIGWSGYSTR(SEQ ID NO：6)

NAIVGVFIKRQY(SEQ ID NO：7)。

conventional antibodies generally refer to tetramers consisting of heavy and light chains of approximately 150kDa in size. Such antibodies are typically obtained by immunizing an animal and then isolating serum or ascites. Therefore, there are often limitations such as poor immune effect, small amount of antibody, lot instability, and high production cost. However, another type of antibody is also found in nature, a dimeric antibody consisting of only heavy chains, called heavy chain antibodies, of about 80-90kDa in size, which is found in camelidae and shark bodies. Meanwhile, previous studies have shown that a single variable region (VHH) portion of a natural heavy chain antibody is sufficient to recognize and bind antigen efficiently, and is about 15kDa in size, simple in structure, and good in stability, and is called a single domain antibody or nanobody. The nano antibody has the advantages of small molecular weight, high antigen affinity, easy folding, good solubility, less modification and easy formation of aggregates, can be produced in a large amount in microorganisms such as bacteria, yeast and the like, and can greatly widen the application of the nano antibody in the basic research field. Therefore, the inventor proposes to develop drosophila protein nanobody, thereby promoting the basic research of drosophila-based model organism to a great extent. According to the invention, drosophila embryo protein lysate is utilized to immunize alpaca, then alpaca peripheral blood lymphocytes are utilized to establish a nano antibody library of drosophila embryo proteins, then purified cyclin E end protein fragments are fixed on the surface of a solid phase carrier, phage display technology is utilized to screen and obtain high-specificity nano antibody genes aiming at the cyclin E proteins for multiple times, and an expression system of the nano antibody in escherichia coli is established, so that the technical effect of continuously producing the cyclin E nano antibody with stable performance on a large scale is realized. The anti-CyclinE nanometer antibody has small molecular weight of about 16kDa, but high antigen binding force and specificity.

In one embodiment, the nanobody has an amino acid sequence as set forth in SEQ ID NO: shown at 8.

The invention also provides a gene for encoding the nano antibody, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: shown at 9.

The invention also provides an expression vector containing the gene.

In one embodiment, the initial vector of the expression vector is a pADL-10b plasmid vector.

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

In one embodiment, the starting strain of the expression strain is E.coli SS320.

The invention also provides a kit for detecting the cyclin E protein, which comprises the nano antibody.

The invention also provides application of the nano antibody in preparation of products for detecting the cyclin E protein.

The invention also provides application of the gene in preparation of products for detecting the cyclin E protein.

Compared with the prior art, the invention has the following beneficial effects:

the anti-CyclinE nanometer antibody has small molecular weight of about 16kDa, but has high antigen binding force and specificity. According to the invention, drosophila embryo protein lysate is utilized to immunize alpaca, then alpaca peripheral blood lymphocytes are utilized to establish a nano antibody library of drosophila embryo proteins, then purified cyclin E end protein fragments are fixed on the surface of a solid phase carrier, phage display technology is utilized to screen and obtain high-specificity nano antibody genes aiming at the cyclin E proteins for multiple times, and an expression system of the nano antibody in escherichia coli is established, so that a foundation is laid for continuous mass production of the cyclin E nano antibody with stable performance.

Drawings

FIG. 1 is an electrophoresis chart in example 1; wherein, FIG. 1A is the nanobody gene electrophoresis chart of example 1, lane M is DNA marker, lane VHH is PCR amplified nanobody gene; FIG. 1B is a colony PCR assay of the quality of nanobody libraries constructed in accordance with the invention in example 1; wherein lane M is a DNA marker, lanes 1 to 16 are monoclonal randomly selected from the constructed nanobody library, and the library insertion rate is detected by colony PCR, and the result shows that the library insertion rate reaches 93.8%;

FIG. 2 is a map of the expression vector pADL-10b-Flag-N3-His in example 4;

FIG. 3 is an electrophoretogram of the nanobody N3 purified in example 4;

FIG. 4 is a graph showing the results of the ELISA method of example 5 for verifying the specific binding of purified nanobody to cyclin E protein;

FIG. 5 is a graph showing the results of plasma resonance technique in example 6 to verify the specific binding and affinity of purified nanobody to CyclinE protein; the curves in FIG. 5A are PBS, cyclin E protein, PBS, N3, and cyclin E protein, N3 from bottom to top; FIG. 5B is a statistical plot of the maximum affinity of each group of specific binding.

Detailed Description

In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The reagents, materials and equipment used in the examples are all commercially available sources unless otherwise specified; the experimental methods are all routine experimental methods in the field unless specified.

Example 1

Constructing drosophila embryo protein nano antibody library.

1. Collecting Drosophila embryo, lysing with lysate to obtain embryo protein lysate, diluting to 1mg/mL, mixing 1mg protein lysate with an equal volume adjuvant (purchased from GERBU Co.) for each immunization, immunizing one alpaca, and immunizing once every two weeks for 4 times.

2. After the 4-time immunization is finished, 50mL of alpaca peripheral blood is extracted, blood lymphocytes are separated, and total RNA of the cells is extracted by a Trizol method.

3. According to PrimerScript from TAKARA Co ^TM 1st Strand cDNA Synthesis Kit, the extracted RNA was reverse transcribed into cDNA and the alpaca heavy chain antibody variable region (i.e., VHH region) was amplified using nested PCR. The primers used for the first round of PCR were as follows:

the upstream primer (5 '-3'): GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO: 10),

downstream primer (5 '-3'): GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 11);

reaction conditions: 98 ℃ for 3min;98 ℃,10s,55 ℃,10s,72 ℃,1min,30 cycles; 72℃for 10min.

The second round of PCR was performed using the first round PCR product as template, using the following primers:

the upstream primer (5 '-3'):

CTCGCGGCCCAGCCGGCCATGGCAGATGTGCAGCTGCAGGAGTCTGGRGGAGG(SEQ ID NO：12)

downstream primer (5 '-3'):

GTGTTGGCCTCCCGGGCCACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT(SEQ ID NO：13)。

reaction conditions: 98 ℃ for 3min;98 ℃,10s,55 ℃,10s,72 ℃,30s,16 cycles; 72℃for 10min.

The fragment VHH of interest with a size of about 500bp was recovered by excision. As shown in FIG. 1A, lane M is a DNA marker, and lane VHH is a VHH fragment, so that a nanobody gene fragment with a size of about 500bp is obtained.

4. 10. Mu.g of phage display vector pADL-10b and 4. Mu.g of VHH were digested with restriction enzyme Bgl II (from NEB Co.) respectively, the two fragments were recovered separately and ligated with T4 ligase (from Thermo Fisher Scientific) ^TM Company) ligates the two fragments and the ligation product is purified.

5. Electrotransformation of ligation products into SS320 competent cells, construction of nanobody bacterial libraries, determination of reservoir capacity, size of about 3X 10 ⁷ And each. Meanwhile, 20 monoclonal antibodies are randomly selected, and the insertion rate of nanobody genes in the established nanobody bacterial library is detected by PCR, wherein the primers are as follows:

the upstream primer (5 '-3'): CAGGAAACAGCTATGACCATGAT (SEQ ID NO: 14),

downstream primer (5 '-3'): GCCCTCATAGTTAGCGTAACGAT (SEQ ID NO: 15).

As a result, as shown in FIG. 1B, about 700bp fragments containing nanobody genes could be amplified from 15 clones among 16 clones picked, indicating that the nanobody bacterial library insertion rate reached 93.8% and the library quality was acceptable.

And obtaining a nanobody phage display library from the nanobody bacterial library by utilizing the helper phage superinfection, and determining the size of the phage library for subsequent screening experiments.

Example 2

Screening of nanobodies against cyclin e proteins.

1. Prokaryotic expression purified the n-terminal 361 amino acid fragment of the cyclin e protein. Mu.g of cyclin E protein was taken with 30. Mu.LDynabeads (Dynabeads) ^TM His-Tag Isolation and Pulldown, invitrogen) was incubated at room temperature for 30min to bind CyclinE to the magnetic beadsThe unbound cyclin E protein was then washed 3 times with PBST.

2. 500. Mu.L phage display library (containing 5X 10) was added to the beads ¹² Phage displaying immune alpaca nanobody), and incubating for 2h at room temperature with turnover. The phage were washed with PBST 25 times to wash off unbound or weakly bound phage. Phage specifically binding to CyclinE was dissociated with 500. Mu.L trypsin (0.25 mg/mL), and 10. Mu. L protease inhibitor cocktail (50X) (available from Roche) was added to the dissociated phage solution for neutralization.

3. 300. Mu.L of the dissociated phage solution was used to infect 3mL of SS320 cells in the logarithmic growth phase, and incubated overnight at 30 ℃. The following day the phage were purified for the next round of screening.

4. This screening procedure was repeated 3 times, and the number of phages used for incubation in the second and third rounds of screening was reduced to 1X 10 ¹² And each.

In the continuous screening process, phage displaying nanobodies specifically binding to cyclene are continuously enriched, and the number of dissociated phage is increased as shown in the following table 1, so that the aim of enriching cyclene nanobodies from the library is fulfilled.

TABLE 1 number of phages before and after screening

Example 3

The enzyme-linked immunosorbent assay (ELISA) screens nanobody positive monoclonal antibodies specifically binding to cyclene.

1. Randomly selecting 20 bacterial monoclonals from the overnight-cultured nanobody bacterial library obtained after the third round of screening, respectively inoculating the bacterial monoclonals into an LB culture medium, culturing until the bacterial growth log phase, adding IPTG with the final concentration of 0.2mM, culturing at 30 ℃ overnight, and inducing nanobody expression.

2. The following day, the cells were collected using CelLytic at 1/10 of the cell volume ^TM B Cell Lysis Reagent (purchased from Sigma Co.) the bacteria were lysed to obtain a crude nanobody extract.100. Mu.L of the crude antibody extract was added to ELISA plates coated with cycline and blocked with 3% BSA and incubated for 1h at room temperature.

3. The unbound proteins were washed off with PBST 5 times, each for 1min. anti-pIII antibody (1:1000, purchased from NEB company) was added and incubated for 1h at room temperature.

4. The unbound antibodies were washed off with PBST 5 times, each for 1min. Alkaline phosphatase-conjugated goat anti-mouse antibody (1:2000, purchased from Abcam corporation) was added and incubated for 1h at room temperature.

5. The unbound antibodies were washed off with PBST 5 times, each for 1min. 200. Mu.L of alkaline phosphatase color development DNPP (purchased from Sigma Co.) was added to each well and developed at room temperature in the dark for no more than 30min.

6. The reaction was terminated by adding 50. Mu.L of 3N sodium hydroxide, and the absorbance was measured on a microplate reader at a wavelength of 405 nm.

7. Clones with an absorbance of 2 times or more that of the negative control group (without nanobody crude body fluid or without anti-pIII antibody) were judged as positive clones. The positive clone bacteria are cultivated in an enlarged mode, plasmids are extracted, and the plasmids are sent to sequencing.

8. The sequencing results were analyzed by comparison using the MegAlign software.

As a result, a DNA sequence with the highest enrichment degree was found, and the gene sequence was designated N3. The nucleotide sequence is shown as SEQ ID NO:9, the amino acid sequence of the corresponding nano antibody is shown as SEQ ID NO:8, the framework region sequence in the amino acid sequence is shown as SEQ ID NO:1-4, the complementarity determining region sequence in the amino acid sequence is set forth in SEQ ID NO:5-7.

MADVQLQESGGGLVQAGGSLRLSCAASG(SEQ ID NO：1)；

FRQAPGKEREFV(SEQ ID NO：2)；

YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC(SEQ ID NO：3)；

WGQGTQVTVSS(SEQ ID NO：4)；

GTFSRYSMGW(SEQ ID NO：5)；

SAIGWSGYSTR(SEQ ID NO：6)；

NAIVGVFIKRQY(SEQ ID NO：7)；

MADVQLQESGGGLVQAGGSLRLSCAASGGTFSRYSMGWFRQAPGKEREFVSAIGWSGYSTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAIVGVFIKRQYWGQGTQVTVSS(SEQ ID NO：8)；

ATGGCAGATGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAGGCACCTTCAGTAGGTATTCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTATCAGCTATTGGATGGAGTGGTTATAGCACACGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGTCTGAAACCTGAAGACACGGCCGTCTATTACTGT

AATGCAATTGTTGGGGTGTTCATCAAACGACAGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGC(SEQ ID NO：9)。

Example 4

Prokaryotic expression and purification of the Cycline nanobody in escherichia coli.

1. The nanobody N3 is cloned into a recombinant vector pADL-10b-Flag-His modified from pADL-10b to obtain an expression vector pADL-10b-Flag-N3-His, the map is shown in figure 2, and the recombinant plasmid is introduced into a host bacterium SS320 for expression by a heat shock method.

2. Selecting monoclonal to LB culture medium containing corresponding antibiotics, culturing at 37deg.C overnight, transferring bacterial liquid to 300mL LB culture medium at 1:100 the next day, and culturing at 37deg.C to OD ₆₀₀ About 0.5, N3 expression was induced by the addition of 0.02% L-arabinose and incubated overnight at 28 ℃.

3. The following day of centrifugation to collect the bacteria, the bacteria were broken by osmotically to obtain crude antibody body fluid, the antibodies were purified by nickel column affinity chromatography, the proteins were washed off with 20mM imidazole, and finally the antibodies were eluted with 250mM imidazole.

4. The obtained nanobody was subjected to electrophoresis, and the result is shown in FIG. 3, wherein lane 1 is a protein marker, and lane 2 is a purified N3 antibody.

Example 5

ELISA method is used for verifying the binding capacity of the cyclin E nano-antibody and cyclin E.

1. First, ELISA was used to perform functional verification on the purified cyclene nanobody, cyclene (1. Mu.g/well) was coated on an ELISA plate, blocked with 3% BSA at 4℃overnight, incubated for 2h at room temperature, and washed with PBST 5 times for 1min each.

2. Nanobody N3 (1. Mu.g/well) was added, incubated at room temperature for 2h, washed 5 times with PBST for 1min each time, and unbound antibody was washed away.

3. Flag antibody (1:1000) was added, incubated at room temperature for 1h, and washed with PBST 5 times for 1min each.

4. HRP conjugated goat anti-mouse antibody (1:2000, purchased from Abcam corporation) was added, incubated for 1h at room temperature, and washed with PBST 5 times for 1min each.

5. Finally, 100 mu L of TMB color development liquid is added, and color development is carried out for 20min at room temperature and in a dark place. The reaction was terminated by adding 50. Mu.L of 2M concentrated sulfuric acid, and the absorbance at a wavelength of 450nm was measured on an ELISA. The experiment set up 2 groups of negative controls, no nanobody added and no HRP-conjugated Flag antibody added, respectively. Each experimental and control group was repeated 3 times.

6. The results show that the experimental groups all exhibited a clear color response and the absorbance results are shown in the following table and fig. 4.

As shown in fig. 4, the ordinate is the absorbance at a wavelength of 450nm, and the abscissa is the experimental group and the negative control group, each value represents 3 independent replicates, and as a result, the absorbance of experimental group N3 is significantly higher than that of the negative control group.

TABLE 2 absorption at 450nm wavelength

Example 6

Plasma resonance technology (SPR) verifies the binding capacity of the CyclinE nanobody to CyclinE.

1. The preparation method comprises the steps of taking a cyclin E protein as a stationary phase, taking a nano antibody as a mobile phase, diluting the stationary phase to a printing concentration by using PBS (phosphate buffer solution) as a stationary phase printing working solution, printing the printing working solution on a 3D photocrosslinking chip by using a BiodotTMAD1520 chip, printing 4 repeated points on each sample by using an array printer, and printing 4 groups of positive control points (Rapamycin) on four corners.

2. Vacuum drying the printed chip, and placing in a photocrosslinking instrument for photocrosslinkingReacting; then DMF, C is used in turn ₂ H ₅ OH，H ₂ O is shaken for 15min, dried by nitrogen, and assembled with a Flowcell Cover for later use. PBST (ph= 7.4,0.1% Tween 20) was added to the mobile phase sample stock, diluted to 5 concentration gradients: 200nM,400nM,800nM,160 nM,3200nM. All samples were circulated in sequence and tested. PBST was used as a flow vehicle throughout the experiment. Interaction test procedure, analyte at 0.5. Mu.L.s ^-1 Flows over the chip surface. In the surface regeneration step, a Glycine-HCl (pH=2.0) solution is used as a regeneration solution, and the flow rate is 2 mu L.s ^-1 。

3. According to the real-time detection result of the SPR equipment, deriving experimental group interaction pairs and negative data, fitting to obtain interaction dynamics curves and outputting affinity parameters.

4. The test results show that the cyclene nanobody has strong binding signals and strong interactions with cyclene proteins, and the curves in FIG. 5A are PBS, cyclene protein, PBS, N3 and cyclene protein, N3 from bottom to top as shown in FIG. 5. Affinity data of SPR test are shown in the following table, and equilibrium dissociation constant of cyclin E protein and cyclin E nanobody is 4.83×10 ^-6 M. Wherein Avg Ka (1/Ms) represents the speed of the binding reaction, a larger Ka represents a faster binding and a smaller Ka represents a slower binding; avgKd (1/s) indicates the rate of dissociation, with a larger Kd representing a faster dissociation and a smaller Kd representing a slower dissociation. Avg KD is an equilibrium dissociation constant, a larger KD indicates more dissociation, representing weaker affinity between ABs, a smaller KD indicates less dissociation, and a stronger affinity between ABs; int. affinity Level is a determination of affinity, 10 ^-13 To 10 ^-8 Is extremely strongly combined with 10 ^-8 Between 10 and 5 is strong binding, 10 ^-5 Up to 2X 10 ^-2 The middle and weak combination is 2 multiplied by 10 ^-2 Very weak bonds between 100. ABS (tr_kd) is an absolute affinity coefficient, ABS (tr_kd) =abs (log 2 (KD)), the higher the value, the higher the affinity.

Table 3 affinity data Profile for SPR analysis of CyclinE and CyclinE nanobodies

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

1. The nanometer antibody of the anti-cyclin E protein is characterized in that the amino acid sequence of the nanometer antibody is shown as SEQ ID NO: shown at 8.

2. A gene encoding the nanobody of claim 1, wherein the nucleotide sequence of the gene is as set forth in SEQ ID NO: shown at 9.

3. An expression vector comprising the gene of claim 2.

4. The expression vector of claim 3, wherein the initial vector of the expression vector is a pADL-10b plasmid vector.

5. An expression strain comprising the expression vector of any one of claims 3-4.

6. The expression strain according to claim 5, wherein the starting strain of the expression strain is E.coli SS320.

7. A kit for detecting a cyclin e protein, comprising the nanobody of claim 1.

8. Use of the nanobody of claim 1 for the preparation of a product for detecting a cyclin e protein.

9. Use of the gene according to claim 2 for the preparation of a product for the detection of a cyclin e protein.