CN108659130B

CN108659130B - Anti-carcinoembryonic antigen nano antibody and application thereof

Info

Publication number: CN108659130B
Application number: CN201810522529.8A
Authority: CN
Inventors: 李文秋; 谢英林; 徐雪松; 王珂; 刘永红
Original assignee: Changchun Lite Biotechnique Co ltd
Current assignee: Changchun Lite Biotechnique Co ltd
Priority date: 2018-05-28
Filing date: 2018-05-28
Publication date: 2021-09-10
Anticipated expiration: 2038-05-28
Also published as: CN108659130A

Abstract

The invention discloses a carcinoembryonic antigen nano antibody, which has unique 3 complementary determining regions CDR1, CDR2 and CDR3, also provides an expression vector containing a coding sequence of a variable region of the nano antibody, a host cell containing the expression vector, a fusion protein of the variable region of the nano antibody and human alkaline phosphatase, an application of the nano antibody in preparing a carcinoembryonic antigen detection kit, a method for carrying out carcinoembryonic antigen immunodetection by applying the nano antibody, and a corresponding detection kit. The anti-CEA nano antibody provided by the invention has specific recognition and binding capacity to CEA, the affinity of the nano antibody can reach 4.791E-10, the nano antibody has a unique antigenic determinant recognition site, and excellent detection effect can be obtained in CEA immunodetection, particularly in a double-antibody sandwich method.

Description

Anti-carcinoembryonic antigen nano antibody and application thereof

Technical Field

The invention discloses a polypeptide, and more particularly discloses an immunoglobulin.

Background

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains 7 domains linked to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor. The 7 domains include a single N-terminal Ig variable domain and 6 domains homologous to Ig constant domains (a1-B1-a 2-B2-A3-B3). CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in several normal adult tissues. Overexpression of CEA is observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers, and therefore CEA has been identified as a tumor-associated antigen. CEA is readily cleaved from the cell surface and shed from tumors into the bloodstream, either directly or via the lymphatic system, and because of this property, serum levels of CEA have been used as clinical markers to diagnose and screen for cancer. Furthermore, CEA has also been used as a tumor marker, and immunological assays to measure elevated CEA in the blood of cancer patients have been used clinically for the prognosis and control of cancer.

More importantly, CEA has become a potentially useful tumor-associated antigen for targeted therapy. There have been reported 2 major approaches to cancer treatment using CEA-targeted immunotherapy: one method uses an anti-CEA antibody to elicit the lytic activity of immune cells, particularly by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEA-expressing tumor cells; another approach is to specifically target CEA-expressing tumor cells by conjugating the anti-CEA antibody or antibody fragment to an effector molecule such as a drug, toxin, radionucleotide, immunomodulator or cytokine, to exert the therapeutic effect of the effector molecule.

Various monoclonal antibodies have been generated against CEA. Chester et al have isolated single chain anti-CEA antibodies from phage display libraries for use in radioimmunoassay and radioimmunotherapy (U.S. Pat. No.5,876,691), followed by humanization of the antibodies (U.S. Pat. No.7,232,888). Radiolabeled anti-CEA antibodies have been used in clinical trials in patients with colorectal cancer.

In 1993, Hamers-Casterman et al found that a class of heavy chain only dimeric antibodies H was found in camelids (camels, dromedary and llamas) in vivo₂It is mainly of the IgG2 and IgG3 type. Such antibodies are lacking a light chain, and thus are referred to as heavy chain-only antibodies (HCabs), and their antigen bindingThe site consists of one domain, called VHH region, and therefore this class of antibodies is also called single domain antibodies or single domain antibodies (sdabs). Since this type of antibody is a variable region sequence after removal of a constant region, the molecular weight is only 15kD, and the diameter is about 10 nm, and thus it is also called nanobody (Nbs). In addition, such single domain antibodies, called VNARs, are also observed in sharks. This heavy chain-only antibody, originally recognized only as a pathological form of human B-cell proliferative disease (heavy chain disease), may be due to mutations and deletions at the genomic level resulting in the inability of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the ability to bind to the light chain, thereby forming a heavy chain dimer.

Nanobodies are comparable in affinity to their corresponding scFv, but surpass scfvs in solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination, relative to scfvs of conventional four-chain antibodies.

The nanobody has the smallest functional antigen-binding fragment derived from HCabs in adult camelids, has high stability and high affinity for antigen binding, and can interact with protein cleft and active sites of enzyme, making its action similar to that of inhibitors. Therefore, the nano-antibody can provide a new idea for designing small molecule enzyme inhibitors from peptide-mimetic drugs. Due to the heavy chain only, nanobodies are easier to manufacture than mabs. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, allow for large yields to be produced at low cost. Therefore, the nano antibody has great value in the treatment and diagnosis of diseases and has great development prospect in the antibody target diagnosis and treatment of tumors.

In view of the fact that CEA is more over-expressed in some solid tumors such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer and thyroid cancer, the development of anti-CEA nanobodies is a new need in the field of antibody technology to fully utilize the super-strong antigen recognition capability of nanobodies, and particularly to recognize some epitopes hidden in crevices or cavities. However, the existence of some structural defects such as low affinity, easy aggregation, short serum half-life and the like due to the low molecular weight of the nanobody prevents the further application of the nanobody. In the specific application of CEA immunoassay, if the antigenic determinants of the CEA recognized by the anti-CEA antibody are too single or the sites are too close or overlapped, the specific antigen-antibody binding reaction is interfered, thereby seriously affecting the detection efficiency. The invention aims to provide an anti-CEA nano antibody which can fully exert the excellent performance of the nano antibody and overcome the inherent defects of the nano antibody, namely, the antibody has high specific antigen recognition capability and affinity, has unique epitope recognition sites, can obtain excellent detection efficiency in the immunoassay of CEA antigen, particularly in a double-antibody sandwich method,

disclosure of Invention

Based on the above objects, the present invention provides a nano-antibody against carcinoembryonic antigen, wherein the variable region of the nano-antibody comprises 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the CDR1 sequence comprises the amino acid sequence shown in SEQ ID No.1, the CDR2 sequence comprises the amino acid sequence shown in SEQ ID No.2, and the CDR3 sequence comprises the amino acid sequence shown in SEQ ID No.3, and the antibody has a unique epitope recognition site.

In a preferred technical scheme, the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID No.4, and a preferable example of the nanobody having the variable region sequence in the present invention is nanobody 2C 5.

Secondly, the invention provides the fusion protein of the nano antibody and the human alkaline phosphatase, the fusion protein is formed by connecting the nano antibody or the variable region fragment thereof and the human placental alkaline phosphatase in series, and in a preferred embodiment of the invention, the sequence of the human placental alkaline phosphatase reporter gene protein is shown as SEQ ID NO. 8.

Thirdly, the invention also provides a nucleotide coding sequence of the nano antibody sequence, and the coding sequence is shown by SEQ ID NO. 5.

Fourthly, the invention provides an expression vector containing the nucleotide coding sequence, and the vector is pMES 4.

Fifth, the present invention provides a host cell containing the above expression vector, wherein the cell is Escherichia coli BL₂₁(DE₃)。

Sixth, the invention also provides the application of the nano antibody in preparing tumor diagnosis reagents.

Seventhly, the invention provides a carcinoembryonic antigen immunodetection method based on non-diagnosis purposes, the method is a double-antibody sandwich enzyme-linked immunoassay method, the variable region sequence of the primary antibody contains an amino acid sequence shown in SEQ ID NO.6, the secondary antibody is an enzyme-linked secondary antibody, and the variable region sequence of the secondary antibody contains an amino acid sequence shown in SEQ ID NO. 4.

In a preferred technical scheme, the enzyme-linked secondary antibody is a fusion protein of a nano antibody of carcinoembryonic antigen and human placental alkaline phosphatase, and in a preferred embodiment of the invention, the sequence of the human placental alkaline phosphatase is shown as SEQ ID NO. 8.

Finally, the invention also provides an immunoassay kit for detecting carcinoembryonic antigen by using a double-antibody sandwich method, the kit comprises a primary antibody for capturing antigen and a secondary antibody for combining with the antigen to initiate an enzyme-linked reaction, the variable region sequence of the primary antibody contains the amino acid sequence shown in SEQ ID NO.6, the secondary antibody is a fusion protein of a nano antibody for resisting carcinoembryonic antigen and human placental alkaline phosphatase, and the fusion protein sequence contains the amino acid sequence shown in SEQ ID NO. 7.

The anti-CEA nano antibody 2C5 provided by the invention has specific recognition and binding capacity to CEA antigen, the affinity of the nano antibody can reach 4.791E-10, and compared with several anti-CEA nano antibodies, the anti-CEA nano antibody recognizes different antigenic determinants and has unique antigenic determinant recognition sites. The fusion protein of the anti-CEA nano antibody and the human alkaline phosphatase provided by the invention not only retains the specific recognition and binding capacity to the CEA antigen, but also introduces the complete activity of the human alkaline phosphatase, and the fusion protein is used as a detection target of a double-antibody sandwich immunoassayThe application of CEA content shows good application prospect, especially unique antigen recognition site, so that the CEA content can be matched with a plurality of other anti-CEA antibodies to be respectively used as a primary antibody and a secondary antibody, the detection efficiency is greatly improved, the detection specificity is ensured, especially the CEA content is matched with a 2C2 nano antibody to be used, a good linear curve is shown, and R represents a good linear curve²0.9916 is reached, approaching a completely linear relationship.

Drawings

FIG. 1 is an SDS-PAGE identification of Nanobody 2C 5;

FIG. 2 is a graph of the affinity test of Nanobody 2C 5;

FIG. 3 is a graph showing the results of cross-reactivity measurement of Nanobody 2C 5;

FIG. 4 is a flow chart of Biacore analysis of Nanobody 2C5 binding site.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

Example 1 construction and screening of anti-CEA Nanobody phage display library

1.1 immunization of alpaca: selecting one healthy adult alpaca, uniformly mixing the recombinant protein CEA and Freund's adjuvant according to the proportion of 1:1, immunizing the alpaca by adopting a back subcutaneous multipoint injection mode according to 6-7 mu g/Kg for four times, wherein the immunization interval is 2 weeks. And collecting alpaca peripheral blood for constructing a phage display library.

1.2 separation of camel source lymphocytes: lymphocytes were analyzed from collected camel-derived anticoagulated whole blood according to routine procedures in the art, every 2.5X 10⁷1mL of RNA isolation reagent was added to each living cell, 1mL of the reagent was extracted with RNA, and the remaining cells were stored at-80 ℃.

1.3 Total RNA extraction: total RNA was extracted according to the routine procedures in the art and adjusted to 1. mu.g/. mu.L with RNase-free water.

1.4 reverse transcription Synthesis of cDNA:

reverse transcription of cDNA was carried out using RNA obtained in 1.3 steps as a template according to the instructions of a reverse transcription KIT (Transcriptor first stand cDNA Synthesis KIT from Roche Co.)

1.5 antibody variable region Gene amplification: and carrying out PCR reaction by using cDNA obtained by reverse transcription as a template. Amplification was performed in two rounds, and the primer sequences for the first round of PCR were as follows:

CALL001：GTCCTGGCTGCTCTTCTACAAGG

CALL002：GGTACGTGCTGTTGAACTGTTCC

the PCR reaction conditions and procedures were: 5min at 95 ℃; 30cycles at 95 ℃ for 30s, 57 ℃ for 30s, 72 ℃ for 30 s; 7min at 72 DEG C

The agarose gel recovery kit gel was used to recover a band of about 700bp, and the nucleic acid concentration was finally adjusted to 5 ng/. mu.l with water.

The primer sequences for the second round of PCR were as follows:

VHH-Back：GATGTGCAGCTGCAGGAGTCTGGRGGAGG

VHH-For：CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

the PCR reaction conditions and procedures were: 5min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 15 cycles; 7min at 72 DEG C

PCR products were purified using a PCR product recovery kit.

1.6 vector construction

Carrying out PstI and BstEII double enzyme digestion on pMES4 and the second PCR product respectively, taking 1.5 mu g of the vector after enzyme digestion and 450ng of the second PCR product after enzyme digestion, adding 15 mu L T4 DNA ligase, supplementing buffer solution and water to the total volume of 150 mu L, carrying out overnight ligation at 16 ℃, and recovering the ligation product. Product recovery was performed using a PCR product recovery kit, eluting with 20. mu.L water.

1.7 electrotransformation and determination of the storage volume

mu.L of the purified ligation product was taken, added to a pre-cooled electric cuvette containing 50. mu.L of E.coli TG1 competent cells, and placed in an electric converter (ECM 630 electric converter of BTX, USA) for electric conversion, and the electric cuvette was taken out, and the transformant was recovered and cultured. Randomly selecting clones, and carrying out colony PCR identification. The pool capacity (pool capacity ═ number of clones × dilution × [ positive rate ] PCR assay × 10) was estimated from the PCR positive rate.

The primer sequences are as follows:

MP57：TTATGCTTCCGGCTCGTATG

GIII：CCACAGACAGCCCTCATAG

1.8 phage amplification

Inoculating recovered bacteria solution into YT-AG culture medium, culturing at 37 deg.C and 200rpm until culture OD₆₀₀0.5. 10ml of the bacterial suspension was taken out and added to 4X 10¹⁰VCSM13, 30min at 37 ℃ for static infection. Centrifuging at 4000rpm at normal temperature for 10min, and removing supernatant. The cells were resuspended in 2 XYT-AK (ampicillin and kanamycin-containing) medium and cultured overnight at 37 ℃ and 200 rpm. Centrifuging, taking a supernatant in a 40ml tube, adding 10ml of PEG/NaCl (20%/2.5M) solution, mixing thoroughly, centrifuging, discarding the supernatant, washing the precipitate with 1ml of ice PBS, centrifuging, taking 250 μ l of precooled PEG/NaCl from the supernatant, mixing thoroughly, washing and resuspending.

Determining the phage titer: TG1 was cultured to OD₆₀₀When the phage was diluted with LB medium in a gradient manner at 0.4, the phage TG1 culture was mixed and cultured in a double dilution manner, and the plaque formation in the plate was observed the next day, and the number of plaques was counted on a dilution gradient plate of 30 to 300 and the phage titer (pfu) was calculated according to the following equation.

Phage titer (pfu/ml) dilution times plaque number times 100

1.9 Nanobody screening

Positive clones were screened for recombinant CEA antigen by ELISA. ELISA plates were coated with recombinant CEA antigen, blocked with 5% BSA, and washed with PBST. Mu.l phage supernatant was added to each well and left at 37 ℃ for 1 h. The supernatant was discarded, and a secondary HRP-labeled mouse anti-M13 antibody was added thereto and the mixture was left at 37 ℃ for 1 hour. Discarding the supernatant, adding TMB solution, incubating at room temperature for 5min, adding 2M sulfuric acid stop solution into each well, and reading with microplate reader at 450 nm.

And selecting the clone with positive phage ELSIA result, and extracting the plasmid. The sequencing results were analyzed using Vector NTI software, and the entries IMGT (see Table II)http://www.imgt.org/IMGT_vquest) Antibody light and heavy chain genes were analyzed to determine the Framework Regions (FRs) and Complementarity Determining Regions (CDRs) of the variable regions.

The nucleotide sequence of the heavy chain of the anti-CEA nano antibody 2C5 is shown as SEQ ID NO.5, the amino acid sequence of the variable region is shown as SEQ ID NO.4, wherein the amino acid sequences at the 1st to 23 th positions are FR1, the amino acid sequences at the 24 th to 31 th positions are CDR1, the amino acid sequences at the 32 th to 48 th positions are FR2, the amino acid sequences at the 49 th to 56 th positions are CDR2, the amino acid sequences at the 57 th to 94 th positions are FR3, the amino acid sequences at the 95 th to 113 th positions are CDR3, and the amino acid sequence at the 114 th position and 124 th positions is FR 4.

Example 2 preparation of anti-CEA Nanobody 2C5

2.1 amplification of original strain TG1 of nano antibody and transformation of Escherichia coli BL21(DE3) by recombinant plasmid of nano antibody

Performing a reaction on an original strain TG1 glycerol strain containing nano antibody nucleic acid according to the ratio of 1: the culture was inoculated at 1000 ratio to 5mL of fresh LB-A medium and cultured overnight at 37 ℃ and 200 rpm. The following day, Plasmid was extracted using a Plasmid mini kit (OMEGA) as per the instructions. After verification, 1. mu.l of the plasmid is transformed into 100. mu.l of competent cells, gently mixed, placed on ice for 30min, hot shocked in a water bath at 42 ℃ for 90s, and cooled in an ice bath for 3 min. 600. mu.l of LB medium was added to the centrifuge tube, and the mixture was cultured with shaking at 37 ℃ for 60 min. 100. mu.l of the supernatant was applied to an LB-A plate using a triangle spreader and cultured overnight at 37 ℃ in an inverted state.

2.2 inducible expression of Nanobodies

The above monoclonal colonies were picked up in LB-A medium and cultured overnight with shaking at 37 ℃. The next day, the bacterial liquid was taken according to the ratio of 1: adding 100ml of fresh LB-A culture medium in a proportion of 100, and performing shaking culture at 37 ℃ for 3h until the bacterial liquid OD₆₀₀After adding IPTG to a final concentration of 1mM, the mixture was induced overnight at 30 ℃. On the third day, 8000rpm, centrifugation for 10min collected the thalli, and 1.5mL of precooled TES buffer was added to resuspend the pellet. After 2min in ice bath, gently shake for 30s and repeat the cycle 6 times. 3.0ml TES/4 (TES diluted 4 times with water) was added, gently shaken for 30s, and then allowed to stand on an ice bath for 2min, and the shaking and standing steps were repeated a total of 6 times. Centrifuging at 9000rpm and 4 deg.C for 10min, and collecting supernatant as periplasm extract.

2.3 purification and characterization of Nanobodies

After resuspending IMAC Sepharose (GE Co., Ltd.), 2ml was taken and added to a gravity column, and the mixture was allowed to stand for 30min to allow Sepharose to naturally settle at the bottom of the gravity column, and the preservation buffer was discharged. Adding 2 times of column volume of nickel sulfate solution (0.1M), and flowing out the nickel sulfate solution at the flow rate of about 8 s/drop; adding 10 times of column volume of balance buffer solution to balance and wash sepharose, and keeping the flow rate unchanged; diluting the sample by 2 times of a balance buffer solution, adding the diluted sample into a gravity column, adjusting the flow rate to be 6 s/drop, and collecting the penetration liquid; adding 10 times of column volume of washing buffer solution to wash sepharose, maintaining the flow rate unchanged, and collecting washing solution; adding elution buffer solution with the volume 3 times of that of the column, maintaining the flow rate at 6 s/drop, and collecting the eluent containing the target protein; finally, the sepharose is washed by sequentially adding 10 times of column volume of the equilibrium buffer, 10 times of column volume of pure water and 10 times of column volume of 20% ethanol, and finally 4ml of 20% ethanol is reserved for storing the chromatographic column. The collected samples were subjected to SDS-PAGE (FIG. 1: M is rainbow 180 broad-spectrum protein Marker; 1 is purified Nanobody 2C 5).

Example 3 affinity Activity of anti-CEA Nanobodies with CEA antigens

3.1 chip antigen coupling

Preparing the CEA antigen into working solution of 20 mu g/mL by using sodium acetate buffer solutions (pH 5.5, pH 5.0, pH 4.5 and pH 4.0) with different pH values, preparing 50mM NaOH regeneration solution, analyzing the electrostatic binding between the antigen and the surface of a chip (GE company) under different pH conditions by using a template method in a Biacore T100 protein interaction analysis system instrument, selecting a proper pH system with most neutral pH according to the standard that the signal increase amount reaches 5 times RL, and adjusting the antigen concentration as the condition during coupling according to the requirement. Coupling the chip according to a template method carried by the instrument: wherein, the 1 channel selects a blank coupling mode, the 2 channel selects a Target coupling mode, and the Target is set as a designed theoretical coupling quantity. The coupling procedure took approximately 60 min.

3.2 analyte concentration setting Condition exploration and regeneration Condition optimization

A manual sample injection mode is adopted, a1, 2-channel 2-1 mode is selected for sample injection, and the flow rate is set to be 30 mu L/min. The sample injection conditions are 120s and 30 mu L/min. Regeneration conditions were all 30s, 30. mu.L/min. The buffer was run continuously empty first until all baselines were stable. The nanobody solution with larger concentration span is prepared to be configured with the running buffer, and 200. mu.g/mL, 150. mu.g/mL, 100. mu.g/mL, 50. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL and 2. mu.g/mL are suggested to be set. Preparing a regeneration solution, selecting the regeneration solution with four pH gradients of a glutamate acid system: 1.5,2.0,2.5,3.0. A200. mu.g/mL sample of analyte was manually injected and the 2-channel was observed, regenerating from the most neutral pH regeneration buffer until the line of response after 2-channel regeneration returned to the same height as the baseline. And manually injecting a sample of 200 mu g/mL of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, regenerating by using a regeneration solution which finally returns the response line to the base line in the previous step, then manually injecting a sample of 200 mu g/mL of analyte once again, observing the signal change of the 2-1 channel and recording the binding capacity, comparing with the value of the previous binding capacity, if the deviation is less than 5 percent, determining that the regeneration solution with the pH value is the optimal regeneration solution, and if the binding capacity of re-injection is lower, continuing to perform the experiment by using a regeneration buffer solution with lower pH value. And taking the selected optimal regeneration solution as a chip surface regeneration reagent after each sample introduction. And respectively injecting analyte concentration samples arranged on the sample injection device, and analyzing the binding capacity of each concentration to finally determine the concentration gradient required by the affinity test.

3.3 affinity assay

According to the optimized sample concentration gradient, the solution is regenerated, and the affinity between the nano antibody and the antigen is tested by using a template method carried by the instrument (wherein the sample introduction condition is set to be 60s and 30 mu L/min; the dissociation time is 600s, and the regeneration condition is set to be 30s and 30 mu L/min). The signal condition of the 2-1 channel is observed at any time. The affinity testing process takes approximately 200 min.

3.4 analysis of results

The binding dissociation curves of a plurality of proper concentration gradients are selected and all the curves are fitted by adopting a 1:1binding mode, and finally important parameters such as affinity values, binding constants, dissociation constants and the like are obtained (figure 2: the binding constants of the nano antibody 2C5 and the antigen CEA are 3.162E +5, and the dissociation constant is 1.515E-4). The affinity value of the anti-CEA nanobody 2C5 was 4.791E-10.

Example 4 Cross-reactogenicity assay for anti-CEA Nanobody 2C5

Mixing antigens CEACAM5, CEACAM1 andCEACAM6 coated the ELISA plates at 0.2 ug/well, incubated overnight at 4 ℃; the next day, 200ul blocking solution (1% BSA) was added to each well, and after incubation for 1h at 37 ℃, PBST was washed 5 times for 3min each; adding 50 μ l of 3 μ g of Nanobody 2C5 into each well, incubating at 37 deg.C for 0.5h, washing with PBST for 5 times, each time for 3 min; using goat anti-alpaca antibody (GE company) marked by HRP as a secondary antibody, and incubating for 0.5h at 37 ℃ according to the dilution ratio of the gradient and 50 ul/hole; adding TMB chromogenic substrate, reacting at room temperature in dark for 10min, adding 2M H₂SO₄The reaction was stopped and the OD read at 450 nm. Negative alpaca serum was also used as a control. The results are shown in fig. 3, where the working concentration of the two antigens CEACAM1 and CEACAM6 reached 1: at 2000, the detected OD value is still lower than that of negative serum, which indicates that the specificity of the nano antibody of the item is good.

Example 5 analysis of Nanobody 2C5 ELISA overlay data

4.1 determination of the saturation concentration of antigen

CEA antigen was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated at 4 ℃ for 24h, and the plate was washed 5 times. Blocking was performed overnight with 1% BSA as blocking agent and the plate was washed 5 times. Adding different gradient diluted nanometer antibodies into the ELISA plate, performing negative control (negative serum 1:100) and PBS blank control, incubating for 30min at 37 ℃, and washing the plate for 5 times. Adding 1: the goat anti-alpaca IgG labeled with HRP diluted at the ratio of 4000 was incubated at 37 ℃ for 30min, and the plate was washed 5 times. Adding TMB developing solution, incubating at 37 ℃ for 10min, and stopping reaction by 2M sulfuric acid. Reading the light absorption value of 450nm, drawing an antibody saturation curve, and selecting the concentration which does not increase with the increase of the concentration as the saturation concentration according to the result.

4.2 site overlay experiments

The first antibody is added for reaction, the second antibody is added after the plate is washed, the enzyme-labeled secondary antibody is added after the plate is washed, and the color reading of TMB is carried out (the method is the same as 4.1). And calculating the overlapping rate AI of the two antibodies, wherein the AI is more than 50 percent, which indicates that the antigenic sites of the 2 antibodies to be detected are different, the AI is less than 50 percent, which indicates that the antigenic epitopes of the two antibodies to be detected are the same, and the larger the AI value is, the lower the possibility of site overlapping is. The formula is as follows: AI [2 a (1+2) - (a1+ a2) ]/a (1+2) × 100%

A1-first Strain antibody reading

A2-second Strain antibody reading

A (1+2) -overlay of 2 antibody readings

Table 1: antibody epitope superposition experiment

	1st antibody	2nd antibody	1st antibody +2nd antibody	Overlap ratio
					2C5+11C12	0.209	0.529	0.642	85.04％
2C5+15E7	0.209	0.466	0.670	99.25％
					2C5+2C2	0.209	0.508	0.772	107.12％

The experimental results are shown in tables 1, 2C5 and 11C12 (the antibody sequence is disclosed in SEQ ID No.1 of CN 106946989A), 15E7 (the antibody sequence is disclosed in antibody VHH-CEA1 of CN106749667A, SEQ ID No.4) and 2C2 (the antibody sequence is disclosed in SEQ ID No.4 of CN 106946989A) three strains of nanobodies respectively aim at different epitopes of CEA, which indicates that in the diagnosis and treatment application of nanobodies, 2C5 nanobodies can be used in combination as a composition for epitope complementation of other nanobodies, so that the diagnosis or treatment efficiency can be increased.

Example 6 analysis of Nanobody 2C5 binding site Using Biacore

The principal principle of the Biacore system is that SPR (refractive index) shifts by changes in the concentration of surface molecules, which appear on the monitor as changes in RU. Due to the higher sensitivity of the system, we designed relevant experiments to verify the ELISA-superimposed experimental results. As shown in fig. 4, first repeating 2 needles of the first nanobody a, observing changes in RU values to confirm saturation of the corresponding antigen binding site and recording; then, a second nanobody B was entered, and RU values were observed and recorded: if the RU value is not more than 20% different from that of the single nano antibody B, the two can be considered to recognize different antigenic determinants; if the difference is more than 20% but less than 60%, the two are considered to have steric hindrance; if the difference value exceeds 60%, the two are judged to recognize the same antigen. The specific operation is that firstly, the increased value R of RU is recorded by the antibody B which is injected only_B1And regenerating the chip; antibody A was then repeated twice and RU increase value R was recorded_AAnd after confirming saturation, directly injecting an antibody B, and observing the increase R of RU value_B2(ii) a Then using the formula (R)_B2-R_A)/R_B1The steric hindrance is calculated to determine whether both recognize the same epitope. The results of this example are shown in Table 2. The steric hindrance of the 2C5 nanobody is 8.94%, 11.38% and 9.15% respectively with three strains of nanobodies, namely 11C12 (the antibody sequence is disclosed in SEQ ID NO.1 of CN 106946989A), 15E7 (the antibody sequence is disclosed in antibody VHH-CEA1 of CN106749667A, SEQ ID NO.4) and 2C2 (the antibody sequence is disclosed in SEQ ID NO.4 of CN 106946989A). Sequentially judging 2C5 and other three strains of nano-antibodiesAll the bodies recognize different antigen sites, and the result is consistent with the result presumed by an ELISA superposition experiment. The application prospect of the anti-CEA nano antibody 2C5 in the field of diagnosis and treatment of CEA is further verified.

Table 2: RU value change table for Biacore detection nano antibody superposition experiment

Example 72 application of C5-HAP to the detection of CEA content in serum of clinical samples

The sequence of the binding site of human placental alkaline phosphatase is taken as the amino acid sequence of a chemical light-emitting region, and the sequence is shown as SEQ ID NO. 8. The flexible polypeptide is fused with the 2C5 nano antibody to form the nano antibody 2C5-HAP with a chemical light-emitting region sequence, and the amino acid sequence of the nano antibody is shown as SEQ ID NO. 7. Two restriction sites HindIII and EcoRI were added to the two ends of the nucleotide coding sequence and ligated to the vector pcDNA3.1 (+). After endotoxin-free large-scale plasmid extraction, 293 cells in logarithmic growth were used for transfection. After the transfected cells are cultured for 36h, the cell culture solution is poured into a 50ml centrifuge tube, 12000g is centrifuged for 5min, the supernatant is collected, filtered by a 0.22um filter membrane, and the culture supernatant is purified by anion exchange chromatography. The affinity test of 2C5-HAP was carried out in the same manner as in example 3, and the affinity value of 2C5-HAP was 8.735E-11. The results of the screening and matching are shown in Table 3. Selecting FC fusion nano antibody 15E7 (the antibody sequence is disclosed in antibody VHH-CEA1 of CN106749667A, SEQ ID NO.4) as a capture primary antibody, the variable region sequence of the FC fusion nano antibody contains the amino acid sequence shown in SEQ ID NO.6, and 2C5-HAP is an enzyme-labeled secondary antibody to detect CEA antigen in a serum sample by a double-antibody sandwich immunoassay method, so that an excellent detection effect is obtained, and the specific process is as follows:

diluting FC fusion nanobody 15E7 with sterile CBS to a final concentration of 10 μ g/ml; adding 100 mul of the enzyme-linked immunosorbent assay (ELISA) plate into each hole, and standing for 18h at 4 ℃; discarding the supernatant, adding 300 μ L of washing solution into each well, shaking horizontally for 3min, and absorbing and discarding the supernatant; the plate was washed four times. Each hole200. mu.l of 1% BSA was added and left to stand at 37 ℃ for 1 h. Washing the plate for four times; adding 50 mu L of positive control, negative control or sample to be detected into each hole; adding 50 mu L of freshly diluted enzyme-labeled secondary antibody (namely nano antibody 2C5-HAP, diluted to the working concentration of 2 mu g/ml) into each hole, and placing the mixture on a shaking table to shake for 3-5 s; incubate at 37 ℃ for 1 h. Washing the plate for four times; adding 100 mu L of AP Chemiluminescence color development liquid (BM Chemiluminescence ELISA Substrate) into each hole, and shaking on a shaking table for 3-5 s; incubating for 10min at room temperature in dark; and selecting a microplate reader program Luminescence, measuring the Lum value of each hole and calculating the CEA value of the quality control serum. Results 2C5 and 15E7 nanometer antibody pair present the best pairing result, positive serum sample is detected as positive, there is no missing detection situation, the average value of positive serum CEA content is 46 ng/ml. And the serum of the CEA sample shows a good linear curve, R^2＝0.9916。

TABLE 3 CEA positive serum detection result of nanometer antibody 2C5-HAP paired with three strains of nanometer antibodies 11C12, 15E7 and 2C2

Sequence listing

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<120> carcinoembryonic antigen nano antibody and application thereof

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Claims

1. An anti-carcinoembryonic antigen nanobody, wherein the variable region of the nanobody has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the CDR1 sequence consists of the amino acid sequence set forth in SEQ ID No.1, the CDR2 sequence consists of the amino acid sequence set forth in SEQ ID No.2, and the CDR3 sequence consists of the amino acid sequence set forth in SEQ ID No. 3.

2. The nanobody of claim 1, wherein the variable region sequence of the nanobody consists of the amino acid sequence set forth in SEQ ID No. 4.

3. The fusion protein of the nanobody of claim 2 and human alkaline phosphatase, wherein the fusion protein is formed by connecting the nanobody and human placental alkaline phosphatase in series.

4. A polynucleotide encoding the nanobody of claim 2, wherein the sequence of the polynucleotide is shown in SEQ ID No. 5.

5. An expression vector comprising the polynucleotide of claim 4, wherein said vector is pMES 4.

6. A host cell comprising the expression vector of claim 5, wherein said cell is E.coli BL21(DE 3).

7. Use of the nanobody of claim 1 or 2 in the preparation of a carcinoembryonic antigen detection kit.

8. The carcinoembryonic antigen immunodetection method based on the non-diagnosis purpose is characterized in that the method is a double-antibody sandwich enzyme-linked immunoassay method, the amino acid sequence of a variable region of a primary antibody is shown as SEQ ID NO.6, a secondary antibody is an enzyme-linked secondary antibody, and the amino acid sequence of the variable region is shown as SEQ ID NO. 4.

9. The method of claim 8, wherein the enzyme-linked secondary antibody is a fusion protein of a nanobody against carcinoembryonic antigen and human placental alkaline phosphatase.

10. An immunoassay kit for detecting carcinoembryonic antigen by using a double-antibody sandwich method comprises a primary antibody for capturing antigen and a secondary antibody for combining with the antigen to initiate an enzyme-linked reaction, and is characterized in that the amino acid sequence of a variable region of the primary antibody is shown as SEQ ID No.6, the secondary antibody is a fusion protein of a nano antibody for resisting the carcinoembryonic antigen and human alkaline phosphatase, and the sequence of the fusion protein is shown as SEQ ID No. 7.