CN116813766B

CN116813766B - High-permeability anti-calmodulin fusion nano-antibody and preparation method and application thereof

Info

Publication number: CN116813766B
Application number: CN202310612515.6A
Authority: CN
Inventors: 王升平; 谭爱梅; 罗帅
Original assignee: Shandong Lifei Biological Industry Co ltd
Current assignee: Shandong Lifei Biological Industry Co ltd
Priority date: 2023-05-29
Filing date: 2023-05-29
Publication date: 2024-02-09
Anticipated expiration: 2043-05-29
Also published as: CN116813766A

Abstract

The application relates to the field of antibody preparation, and particularly discloses a high-permeability anti-calmodulin fusion nano-antibody, a preparation method and application thereof. High permeability anti-calmodulin fusion nanobodies include anti-calmodulin nanobodies that include a single domain heavy chain variable region that includes a CDR-L1, CDR-L2, and CDR-L3 that include a particular amino acid sequence. The preparation method comprises the following steps: preparing recombinant calmodulin; immunizing alpaca by using recombinant calmodulin as an antigen, and constructing a phage library by using peripheral blood lymphocytes of the immunized alpaca; panning the phage library to obtain the anti-calmodulin nano-antibody. The high-permeability anti-calmodulin fusion nano-antibody can be used for in-vitro diagnosis calmodulin immunohistochemical detection, and has the advantages of high tissue penetrability, high specificity and high affinity.

Description

High-permeability anti-calmodulin fusion nano-antibody and preparation method and application thereof

Technical Field

The application relates to the field of antibody preparation, in particular to a high-permeability anti-calmodulin fusion nano-antibody, a preparation method and application thereof.

Background

Nanobodies, or single domain heavy chain antibodies, comprise only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, lack light chains as compared to conventional antibodies, and have no CH1 region between the heavy chain variable region and the hinge region, with a relative molecular mass of less than 15kDa, being the smallest unit currently known to bind an antigen of interest. The nano antibody can be combined with antigen with high specificity and high affinity, has strong tissue penetrating power and high stability, and has wide application prospect in the aspects of diagnosis and treatment of diseases.

Calponin is a calmodulin that binds tropomyosin and F-actin, a specific protein of smooth muscle cells, and has the function of regulating smooth muscle contraction. Calponin is a differentiation marker protein, is abundantly expressed in differentiated smooth muscle cells, and is involved in actin cytoskeletal reconstruction by binding to cytoskeletal protein actin, and has various biological functions, and changes in expression are closely related to the occurrence and development of many diseases.

Clinically, malignant solid tumor patients requiring Calponin immunotherapeutic drugs need to be subjected to immunohistochemical detection of tumor tissues in advance to determine whether the tumor tissues express Calponin molecules and the cell proportion of the Calponin, so that anti-Calponin drugs are used more pertinently, doctors and patients are assisted in reasonably selecting treatment modes, and the penetration capability of antibodies for detection is high due to the specificity of immunohistochemical detection samples. At present, most antibodies for in vitro diagnosis calmodulin immunohistochemical detection are murine monoclonal antibodies, however, the relative molecular weight of the murine monoclonal antibodies is about 150kDa, the volume is larger, the membrane penetrating capacity and the tissue penetrating capacity are weaker, and the sensitivity and the accuracy of detection are deficient.

In view of the above related art, the inventors believe that the conventional murine monoclonal antibodies have a disadvantage of poor tissue penetration when used in calmodulin immunohistochemical detection.

Disclosure of Invention

In order to improve the tissue penetrability of an antibody for in vitro diagnosis calmodulin immunohistochemical detection, the application provides a high-permeability anti-calmodulin fusion nano-antibody, a preparation method and application thereof.

In a first aspect, the present application provides a high-permeability anti-calmodulin fusion nanobody, which adopts the following technical scheme:

a high permeability anti-calmodulin fusion nanobody comprising an anti-calmodulin nanobody comprising a single domain heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences:

CDR-H1：LIFEIYSSA；CDR-H2：VKGR；CDR-H3：ARNPTG。

preferably, the single domain heavy chain variable region comprises the amino acid sequence shown below:

QVQLQESGGGSGQVGTSLRLSCLIFEIYSSACMGWFRQAPGKEREWVHGSIVSFSDARTNYADAVKGRFTGTQDNAKNTVYLQMNSLKPEDTAMYYCARNPTGINKTRRYDYWGQGTQVTVSS。

by adopting the technical scheme, the anti-calmodulin nano-antibody is specifically combined with calmodulin, has small molecular weight and higher tissue penetrability, has good adaptability to samples, can enable intracellular and intra-tissue imaging to be clearer and more accurate, and is more suitable for calmodulin immunohistochemical detection;

in vivo half-life of the anti-calmodulin nano antibody is controllable, in tumor treatment, as normal smooth muscle cells also express Calponin in a low amount, and the requirement of the anti-calmodulin nano antibody drug for controlling apoptosis of tumor cells by tumor microenvironment on half-life is very strict, the anti-calmodulin nano antibody with controllable half-life can be more suitable for diagnosis of tumors;

the anti-calmodulin nano-antibody has extremely high homology with human body and small molecular weight, so that the extremely low rejection can be achieved by carrying out a small amount of humanization on the anti-calmodulin nano-antibody, thereby being beneficial to reducing the difficulty of antibody drug humanization.

Preferably, the high permeability anti-calmodulin fusion nanobody further comprises horseradish peroxidase in tandem with the anti-calmodulin nanobody.

By adopting the technical scheme, the anti-calmodulin antibody is connected with HRP (horseradish peroxidase) in series, the target protein can be directly positioned in the immunohistochemical detection, the second antibody incubation step is reduced, the one-step immunohistochemical detection is realized, the detection time is reduced, the possibility of introducing non-specific binding in the second antibody incubation step can be reduced, and the accuracy of the detection result is improved.

In a second aspect, the present application provides a method for preparing a high-permeability anti-calmodulin fusion nanobody, which adopts the following technical scheme:

the preparation method of the high-permeability anti-calmodulin fusion nano-antibody comprises the following steps:

preparing recombinant calmodulin;

immunizing alpaca by using recombinant calmodulin as an antigen, and constructing a phage library by using peripheral blood lymphocytes of the immunized alpaca; panning the phage library to obtain the anti-calmodulin nano-antibody.

By adopting the technical scheme, the anti-calmodulin nano-antibody with high specificity, high affinity and high permeability can be prepared.

Preferably, the method for preparing recombinant calmodulin comprises the steps of:

the calmodulin gene is obtained, cloned to a recombinant vector, host cells are transformed, recombinant calmodulin is expressed, and the recombinant calmodulin is purified.

Preferably, the recombinant calmodulin is purified by nickel column affinity chromatography.

Preferably, the method of immunizing alpaca with recombinant calmodulin as antigen comprises:

injecting 0.1-0.5mg of recombinant calmodulin into alpaca body for animal immunization by adopting a subcutaneous multipoint injection mode at the neck and back, wherein an immunization adjuvant is squalene adjuvant;

multiple immunization injections were performed, each at 20-30 days intervals.

Preferably, the step of constructing a phage library comprises:

extracting RNA of peripheral blood lymphocytes of the immunized alpaca, and carrying out reverse transcription to obtain cDNA;

performing PCR amplification by taking a cDNA chain as a template to obtain a heavy chain variable region gene fragment of the anti-calmodulin, connecting the heavy chain variable region gene fragment of the anti-calmodulin into a display carrier, transforming competent cells, and constructing a nanobody library;

infecting the nano antibody library with phage to obtain phage display gene library, and panning by phage display technology to obtain the anti-calmodulin nano antibody.

Preferably, the PCR amplification is nested PCR, comprising the steps of:

performing first PCR amplification by taking the cDNA chain as a template;

carrying out 2% agarose gel electrophoresis on the product amplified by the first PCR, and recovering 600-800bp bands;

and carrying out a second PCR amplification by taking the product recovered by the first PCR glue as a template.

By adopting the technical scheme, the amplification specificity is improved, and the specificity of the obtained anti-calmodulin heavy chain variable region gene is improved.

Preferably, the display vector is a pMES4 vector and the competent cells are TG1 competent cells.

In a specific embodiment, the method of preparation further comprises the steps of:

obtaining a single domain heavy chain variable region gene sequence of the anti-calmodulin antibody through gene sequencing;

obtaining horseradish peroxidase gene sequences;

and (3) connecting the single-domain heavy chain variable region gene sequence of the anti-calmodulin antibody with the horseradish peroxidase gene sequence in series, cloning into an expression vector, constructing a recombinant expression vector, transfecting a host cell with the recombinant expression vector, expressing the anti-calmodulin fusion nanobody, and purifying the anti-calmodulin fusion nanobody.

Preferably, the expression vector is a PCDNA3.1 (+) vector and the host cell is a HEK293 cell.

Preferably, the anti-calmodulin fusion nanobody is purified using an immunoaffinity chromatography column.

In a third aspect, the present application provides an application of a high-permeability anti-calmodulin fusion nanobody, which adopts the following technical scheme:

the application of the high-permeability anti-calmodulin fusion nano-antibody is applied to in-vitro diagnosis calmodulin immunohistochemical detection.

In a specific embodiment, the application comprises the steps of:

preparing tissue slices and performing serum sealing;

dripping the anti-calmodulin fusion nano-antibody on a tissue slice, and incubating;

color development, washing, counterstain, differentiation, blue reflection, dehydration, sealing and microscopic observation.

By adopting the technical scheme, as the anti-calmodulin fusion nanobody can position the target protein, the secondary antibody incubation step is reduced, the time of immunohistochemical detection is shortened, the one-step immunohistochemical detection is realized, the possibility of introducing non-specific binding in the secondary antibody incubation step is reduced, and the accuracy of imaging is improved; because the anti-calmodulin fusion nano antibody has smaller molecular weight and excellent tissue penetrability, the anti-calmodulin fusion nano antibody has better adaptability to tissue slice samples, the imaging is clearer and more accurate, the accuracy of detection results is improved, and the accuracy and the sensitivity of diagnosis and detection of various diseases such as tumors are improved.

In a fourth aspect, the present application provides an application of a high-permeability anti-calmodulin fusion nanobody, which adopts the following technical scheme:

a calmodulin detection kit comprising the high-permeability anti-calmodulin fusion nanobody.

By adopting the technical scheme, the calmodulin detection kit with high detection accuracy and high sensitivity can be obtained.

In summary, the present application has the following beneficial effects:

1. the anti-calmodulin fusion nano-antibody has the advantages of small molecular weight, excellent tissue penetrability, good adaptability to tissue samples and strong affinity, can enable intracellular and intra-tissue imaging to be clearer and more accurate, is more suitable for calmodulin immunohistochemical detection, and is beneficial to improving the accuracy of detection results; the half-life in vivo is controllable, so that the method is more suitable for diagnosing and monitoring tumors; meanwhile, the homology with the human body is extremely high, which is beneficial to reducing the difficulty of humanization of antibody medicaments;

2. the anti-calmodulin fusion nano antibody can realize one-step immunohistochemical detection, shortens detection time, reduces the possibility of nonspecific binding introduced by a secondary antibody imparting step, improves the accuracy of imaging, and further improves the accuracy of detection results.

Drawings

FIG. 1 is a nucleic acid electrophoresis result of Calponin-PCDNA3.1 (+) vector in example 1 of the present application;

FIG. 2 shows the results of a monoclonal plating of TOP10 competent cell Calponin-PCDNA3.1 (+) vector transformed in example 1 of the present application;

FIG. 3 is a SDS-PAGE reduction electrophoresis of Calponin recombinant protein expression in HEK293 cells of example 1 of the present application;

FIG. 4 shows the results of nucleic acid electrophoresis of colony PCR products in example 1 of the present application;

FIG. 5 is a library capacity measurement 10 of the anti-Calponin nanobody library of example 1 of the present application ⁸ Plating results after dilution;

FIG. 6 is a result of nucleic acid electrophoresis of HRP-PCDNA3.1 (+) vector in example 1 of the present application;

FIG. 7 is a plating result after TOP10 competent cells were transformed with HRP-PCDNA3.1 (+) vector in example 1 of the present application;

FIG. 8 is a result of nucleic acid electrophoresis of the VHH-HRP-PCDNA3.1 (+) vector of example 1 of the present application;

FIG. 9 is the plating results after transformation of TOP10 competent cells with VHH-HRP-PCDNA3.1 (+) vector in example 1 of the present application;

FIG. 10 shows the result of SDS-PAGE reduction electrophoresis of a cell broth of HEK293 cells transformed with VHH-HRP-PCDNA3.1 (+) vector according to example 1 of the present application;

FIG. 11 is a microscopic examination of the staining effect of anti-Calponin fusion nanobody on paraffin-embedded tissue sections in example 2 of the present application;

FIG. 12 is a microscopic examination of the staining effect of murine anti-Calponin antibodies on paraffin-embedded tissue sections in the comparative example of the present application.

Detailed Description

The present application is described in further detail below with reference to the drawings and examples.

Examples

Example 1

(one) preparation of Calponin recombinant protein

Calponin gene (accession No. nm_ 001299.6) was synthesized by gene company as follows: ATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGAGCCTGGCTCTGGTGACCAACTCTATGAGCAGCGCCCACTTCAACAGGGGCCCTGCTTACGGACTGAGCGCTGAAGTGAAGAACAAGCTGGCCCAGAAATACGACCACCAGAGAGAACAGGAGCTGAGAGAGTGGATCGAGGGCGTGACAGGAAGAAGAATCGGAAATAACTTTATGGACGGACTGAAGGACGGAATCATCCTGTGTGAGTTCATCAACAAGCTGCAGCCAGGAAGCGTGAAGAAGATCAACGAGAGCACACAGAACTGGCACCAGCTGGAGAACATCGGGAACTTCATTAAAGCCATCACCAAGTACGGCGTGAAGCCCCACGACATCTTCGAGGCTAACGACCTGTTCGAGAACACAAACCACACCCAGGTGCAGAGCACACTGCTGGCTCTGGCTAGCATGGCCAAGACCAAGGGCAACAAGGTGAACGTGGGCGTGAAATACGCCGAGAAACAGGAGAGAAAGTTCGAGCCCGGCAAGCTGAGAGAGGGAAGAAACATCATCGGACTGCAGATGGGCACCAACAAATTCGCCAGCCAGCAGGGAATGACAGCCTACGGAACCAGAAGACACCTGTACGACCCCAAGCTGGGAACCGACCAGCCTCTGGATCAGGCTACCATCAGCCTGCAGATGGGAACCAACAAAGGAGCCAGCCAGGCCGGAATGACCGCTCCTGGAACAAAAAGACAGATCTTCGAACCCGGCCTGGGAATGGAGCACTGCGATACACTGAACGTGTCACTGCAGATGGGGAGCAACAAGGGCGCCTCACAGAGAGGAATGACCGTGTACGGACTGCCCAGACAGGTGTACGACCCCAAATACTGTCTGACCCCTGAGTACCCCGAACTGGGGGAACCTGCTCACAATCACCACGCCCACAACTACTACAACAGCGCCCACCATCACCATCACCAT.

The Calponin gene is cloned onto PCDNA3.1 (+) vector by adopting homologous recombination technology, the Calponin-PCDNA3.1 (+) vector is detected, the detection result is shown in figure 1, wherein the target band of the empty vector is 5428bp, the band of the Calponin gene fragment is 969bp, and the band of the Calponin-PCDNA3.1 (+) vector is 6397bp after construction, which indicates that the Calponin-PCDNA3.1 (+) vector is constructed successfully;

TOP10 competent cells were transformed and subjected to monoclonal plating, and the results are shown in FIG. 2;

selecting positive clones, culturing cells, extracting Calponin-PCDNA3.1 (+) vector plasmids, carrying out gene transfection on HEK293 cells, carrying out shake table expression for 5 days by adopting a serum-free culture medium, carrying out high-speed centrifugation, taking supernatant to obtain cell fermentation liquid, carrying out protein electrophoresis analysis on the cell fermentation liquid, and the analysis result is shown as figure 3, wherein the HEK293 cells can successfully express Calponin recombinant protein, and the purity is more than or equal to 95%.

Calponin recombinant protein was purified using nickel column affinity chromatography.

(II) immunization of animals

Injecting Calponin recombinant protein into three alpaca bodies for animal immunization by adopting a subcutaneous multipoint injection mode at the neck and back; four immunization injections of 0.3 mg/time of Calponin recombinant protein are respectively carried out on each alpaca, the immunization adjuvant is squalene adjuvant, and each immunization interval is 21 days.

After the fourth immunization, ELISA titers of the peripheral blood of three immunized alpacas and the peripheral blood of the non-immunized alpacas were collected respectively, and the detection results are shown in Table 1:

table 1 immune titer assay

Referring to table 1, it can be seen that alpaca produced excellent immune titers after immunization.

(III) construction of phage library

3.1 obtaining VHH Gene fragments against calmodulin

Collecting 20mL of peripheral blood of jugular vein of the immune alpaca 1, separating peripheral blood lymphocytes, extracting total RNA, and performing reverse transcription to obtain cDNA;

the first PCR amplification was performed using cDNA strand as template, and the primer sequences were as follows:

CALL001	5’-GTCCTGGCTGCTCTTCTACAAGG-3’
		CALL002	5’-GGTACGTGCTGTTGAACTGTTCC-3’

electrophoresis was performed using a gel with an agar powder content of 2%, a band of 650-750bp was recovered, and the final nucleic acid concentration was adjusted to 5 ng/. Mu.L with water.

And (3) taking the product recovered by the first PCR glue as a template, and carrying out second PCR amplification, wherein the primer sequences are as follows:

VHH-Back	5’-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3’
		VHH-For	5’-CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3’

purifying the PCR product using Pure Cycle kit (OMEGA) to obtain a gene fragment comprising VHH (single domain heavy chain variable region) of anti-Calponin nanobody;

3.2 construction of anti-Calponin nanobody library

Double digestion of phage vector pMECS with restriction enzyme PstI-HF and BstEII-HF; double digestion treatment is carried out on the purified secondary PCR product by using restriction endonucleases PstI-HF and BstEII-HF, 1.5% agarose gel electrophoresis is carried out on the digested product, 5000bp carrier bands and 400-500bp VHH bands are cut off after electrophoresis, and Gel Extraction Kit is used for recovery;

phosphorylating the recovered pMES4 vector, and recovering the treated nucleic acid using the Cycle Pure Kit (OMEGA); the VHH gene fragment was ligated into pMES4 vector using T4 DNA ligase, and each 50. Mu.L ligation system included 0.35. Mu.g of vector after cleavage, 0.1. Mu.g of VHH gene fragment after cleavage, 5.0. Mu.L of T4 DNA ligase, 10×T4Buffer5.0. Mu.L, and the balance sterile water. The ligation was carried out overnight at 16℃and the product was recovered using the Cycle Pure Kit and finally eluted with 20. Mu.L of deionized water.

10. Mu.L of the purified ligation product was added to 100. Mu.L of TG1 competent cells for electrotransformation at 2.5kV,200Ω and 2.5. Mu.F. After electrotransformation, 1mL of 2 XYT culture solution preheated at 37 ℃ is added, and shaking recovery culture is carried out for 1.5h at 37 ℃ and 200 rpm; diluting the resuscitated bacterial liquid to 10 in gradient ⁴ Multiple of 10 ⁵ Multiple of 10 ⁶ Multiple of 10 ⁸ 100. Mu.L of each was plated on LB-AG solid plates and incubated overnight at 37℃and all colonies were collected the next day to obtain an anti-Calponin nanobody library.

3.3 library Capacity assays against Calponin nanobody library

Selecting a transformed and revived TG1 competent cell monoclonal, transferring the monoclonal into 10 mu L of water, and uniformly mixing to obtain a colony test solution;

colony test solution was taken and colony PCR was performed with the following PCR primers:

pMES-F	GCC GCT GGA TTG TTA TTA CTC
		pMES-R	CTT TCA ACA GTG GAA CCG TAG

the PCR products were electrophoresed using a gel with an agar powder content of 1.5%, and the results are shown in FIG. 4, which indicate that the VHH gene of the anti-Calponin nanobody can be successfully expressed. According to the electrophoresis result, the PCR positive rate is calculated to be 70.5%.

Dilution 10 ⁸ The results of cell culture of the bacterial solution after double transformation and recovery are shown in FIG. 5, 17 single colonies are obtained, and the library capacity of the anti-Calponin nanobody library is calculated to be 17×10 according to the library capacity=clone number×dilution number×positive rate×10 ⁸ ×70.5％≈1.9×10 ⁹ 。

3.4 construction of anti-Calponin nanobody phage display library

Taking 500 mu L of the resuscitated bacterial liquid after electrotransformation, inoculating the bacterial liquid into 50ml of 2 XYT-AG culture medium, and shake culturing at 37 ℃ and 200rpm until the OD600 of the logarithmic phase is 0.5;

10ml of the bacterial liquid is taken out and added with 4 multiplied by 10 ¹⁰ phage VCSM13 of pfu, resting at 37℃for 30min; after centrifugation, the supernatant was removed, and the cells were resuspended in 2 XYT-AK medium and shake-cultured overnight at 37℃and 200 rpm. Centrifuging on the next day, taking 40ml of supernatant, adding 10ml of PEG8000/NaCl (20%/2.5M) solution, uniformly mixing, ice-bathing for 1h, centrifuging, removing supernatant, re-suspending with 8ml of pre-cooled PBS, adding 2ml of pre-cooled PEG/NaCl into the re-suspension, ice-bathing for 20min, centrifuging, re-suspending with 1ml of pre-cooled PBS, re-centrifuging, and taking supernatant to obtain the anti-Calponin nanobody phage display library.

3.5 phage panning

Calponin recombinant protein was diluted to 10. Mu.g/ml with PBS, and 100ul per well was plated in ELISA plates overnight at 4 ℃. The PBST is washed 3 times in the next day, 250 μl of 5% BSA is added, and the mixture is blocked for 2 hours at 37 ℃; blocking solution was discarded, washed 5 times with PBST, and 90. Mu.l phage display library (10 ¹¹ cfu/well) and 10 μl of 5% BSA blocking solution, incubated at 37deg.C for 2h, PBST washed 15 times; after that, 100. Mu.l of 0.2M Gly (pH 2.2) was added to each well, incubated at room temperature for 15min on a horizontal shaker, and the eluate was transferred to a centrifuge tube into which 15ul of 1M Tris (pH 9.1) had been previously added.

Mu.l of the above eluate was added to 3ml of TG1 competent cells having an OD600 of 0.5, and the mixture was subjected to stationary culture at 37℃for 30min, 7ml of LB, 100. Mu.g/ml of ampicillin and 2% of glucose were added thereto, and the mixture was subjected to shaking culture at 200rpm at 37℃overnight.

Inoculating the above cultured bacteria at 1:100 into 50ml of 2YT-AG culture medium, culturing at 37deg.C and 200rpm until OD600 = 0.5, and adding 10ml of bacterial liquid into 4×10 ¹⁰ Standing pfu phage VCSM13 at 37deg.C for 30min, centrifuging, removing supernatant, adding 50ml 2YT-AK, suspending, shake culturing at 37deg.C at 200rpm overnight, and precipitating phage the next day;

the second round of screening was performed, which differs from the first round of screening in that: the recombinant protein coating amount was adjusted to 2ug/ml, the phage washing times were increased to 20 times, and the tween-20 concentration in the PBST for washing was increased to 0.1%.

Through multiple rounds of screening, positive clones are continuously enriched, so that the aim of screening Calponin specific nano antibodies in an antibody library by utilizing a phage display technology is fulfilled.

(IV) obtaining the VHH gene sequence of the anti-calmodulin nano-antibody

4.1 phage ELISA screening

Taking TG1 competent cells cultured overnight after screening, diluting to 10 by 10-fold gradient with LB culture medium ⁵ Double sum 10 ⁶ 100ul of each was plated on LB-AG plates and incubated overnight at 37 ℃. The following day, from the plates, single clones were selected into 96-well plates containing 100ul of 2YT-AG medium (10% glycerol) and incubated at 37℃overnight; from the 96-well plate, 172 clones were inoculated at 1:50 into 250. Mu.l of 2YT-AG medium, shake-cultured at 37℃and 200rpm for 3 hours, and 1ul of helper phage (10 were added ⁹ pfu), stationary incubation at 37℃for 30min, centrifugation to remove supernatant, adding 1ml of 2YT-AK to resuspend pellet, shake cultivation at 200rpm at 37℃overnight. The following day, the phage supernatant was removed by centrifugation.

Calponin recombinant protein was diluted to 5. Mu.g/ml with PBS and 100ul per well was plated in ELISA plates overnight at 4 ℃. The following day, ELISA plates were blocked with 5% BSA, washed with PBST after blocking, 100ul phage supernatant was added to each well, incubated for 1h at 37℃and the supernatant was discarded and PBST washed 3 times. HRP-labeled murine anti-M13 secondary antibody was diluted 1:50000 with PBS and added to the plate at 100ul per well and incubated for 1h at 37 ℃. After 3 times of PBST washing, 100ul of TMB solution was added to the plate per well, incubated at room temperature in the dark for 15min, 100ul of 2M sulfuric acid stop solution was added per well, and read with an ELISA reader at 450 nm. The ELISA results for the two 96 plates are shown in table 2:

TABLE 2 phage ELISA results

4.2 Gene sequencing

According to Table 2, it can be seen that the clones of row 1A, column 9 of the second plate (clone numbers 2-1A 9) have the highest OD and highest reaction affinity.

anti-Calponin nanobody clone No. 2-1A9 was sequenced and the VHH region was sequenced as follows:

IMGT analysis of the sequences of anti Calponin nanobodies gave the following CDR regions:

CDR	sequence(s)
		CDR1	LIFEIYSSA
CDR2	VKGR
		CDR3	ARNPTG

(V) construction of anti-calmodulin fusion nanobody

5.1 construction of expression vectors

The HRP gene (Gene accession number: J05552.1) was synthesized from the genetic idea and the gene sequence was as follows:

ATGCAGTTAACCCCTACATTCTACGACAATAGCTGTCCCAACGTGTCCAACATCGTTCGCGACACAATCGTCAACGAGCTCAGATCCGATCCCAGGATCGCTGCTTCAATATTACGTCTGCACTTCCATGACTGCTTCGTGAATGGTTGCGACGCTAGCATATTACTGGACAACACCACCAGTTTCCGCACTGAAAAGGATGCATTCGGGAACGCTAACAGCGCCAGGGGCTTTCCAGTGATCGATCGCATGAAGGCTGCCGTTGAGTCAGCATGCCCACGAACAGTCAGTTGTGCAGACCTGCTGACTATAGCTGCGCAACAGAGCGTGACTCTTGCAGGCGGACCGTCCTGGAGAGTGCCGCTCGGTCGACGTGACTCCCTACAGGCATTCCTAGATCTGGCCAACGCCAACTTGCCTGCTCCATTCTTCACCCTGCCCCAGCTGAAGGATAGCTTTAGAAACGTGGGTCTGAATCGCTCGAGTGACCTTGTGGCTCTGTCCGGAGGACACACATTTGGAAAGAACCAGTGTAGGTTCATCATGGATAGGCTCTACAATTTCAGCAACACTGGGTTACCTGACCCCACGCTGAACACTACGTATCTCCAGACACTGAGAGGCTTGTGCCCACTGAATGGCAACCTCAGTGCACTAGTGGACTTTGATCTGCGGACCCCAACCATCTTCGATAACAAGTACTATGTGAATCTAGAGGAGCAGAAAGGCCTGATACAGAGTGATCAAGAACTGTTTAGCAGTCCAAACGCCACTGACACCATCCCACTGGTGAGAAGTTTTGCTAACTCTACTCAAACCTTCTTTAACGCCTTCGTGGAAGCCATGGACCGTATGGGTAACATTACCCCTCTGACGGGTACCCAAGGCCAGATTCGTCTGAACTGCAGAGTGGTCAACAGCAACTCT。

by adopting the homologous recombination technology, the HRP gene is seamlessly cloned to the PCDNA3.1 (+) vector to obtain the HRP-PCDNA3.1 (+) vector, the construction result is detected by nucleic acid electrophoresis, the electrophoresis result is shown in figure 6, and the 900bp HRP gene is successfully cloned into the vector to obtain the HRP-PCDNA3.1 (+) vector. TOP10 competent cells were transformed with HRP-PCDNA3.1 (+) vector, and the results are shown in FIG. 7, which shows that the vector transformation capacity was strong.

The HRP-PCDNA3.1 (+) vector is subjected to double digestion treatment by using restriction endonuclease EcoRI, handIII, the anti-Calponin nanobody VHH gene of clone number 2-1A9 is connected to the HRP-PCDNA3.1 (+) vector by using T4 DNA ligase to obtain the VHH-HRP-PCDNA3.1 (+) vector, the construction result is detected by nucleic acid electrophoresis, and the electrophoresis result is shown in FIG. 8, and the anti-Calponin nanobody gene is successfully connected to the vector to obtain the VHH-HRP-PCDNA3.1 (+) vector.

TOP10 competent cells were transformed with VHH-HRP-PCDNA3.1 (+) vector, as shown in FIG. 9, with better transformation results.

5.2 expression of anti-Calponin fusion nanobodies

Positive monoclonal of TOP10 competent cells transformed by VHH-HRP-PCDNA3.1 (+) vector is selected, inoculated into 5ml of LB-AG culture medium at a ratio of 1:100, shake cultured overnight at 37 ℃ and 200rpm, plasmids are extracted, HEK293 cells are transfected by genes, the transfected HEK293 cells are expressed for 5 days in a serum-free culture medium table, the supernatant is centrifuged, cell fermentation liquid of the supernatant is taken, and the result is shown as figure 10, and the anti-Calponin fusion nanobody can be successfully expressed, and the purity is more than or equal to 95%.

The anti-Calponin fusion nanobody was purified using VHH immunoaffinity chromatography column.

Example 2

An application of a high-penetrability anti-calmodulin fusion nano-antibody, which comprises the following steps:

taking paraffin embedded tissue slices, putting the paraffin embedded tissue slices into serum with the same species source as the anti-Calponin fusion nano antibody, sealing at room temperature, and removing sealing liquid after sealing is finished;

diluting the anti-Calponin fusion nanobody to 1 mug/mL, dripping the anti-Calponin fusion nanobody on a tissue slice until the anti-Calponin fusion nanobody covers the tissue, incubating the tissue slice for 1h at 37 ℃, and flushing the tissue slice with PBST for 5 times;

dripping DAB for color development for 5min, and flushing with ultrapure water for 10min;

counterstaining with hematoxylin dye for 15s, and flushing with ultrapure water until the water is clear;

differentiation treatment is carried out for 3s by 1% hydrochloric acid, and then the mixture is washed by ultrapure water for 5 times;

placing the tissue slice treated by the method into a lithium carbonate bluing solution to be blued for 2s, and then flushing the tissue slice with ultrapure water for 15min;

the pellet was dehydrated and sealed, and the result was observed under a microscope, as shown in FIG. 11.

Comparative example

This comparative example differs from example 2 in that immunohistochemical monitoring was performed using murine anti-Calponin antibodies. The method comprises the following specific steps:

taking paraffin embedded tissue slices, putting the paraffin embedded tissue slices into serum with the same species source as a murine anti-Calponin antibody, sealing at room temperature, and removing sealing liquid after sealing is finished;

the murine anti-Calponin antibody was diluted to 1 μg/mL, added dropwise to the tissue sections, the tissue sections were incubated for 1h at 37 ℃ and washed 5 times with PBST;

dripping HRP-marked goat anti-mouse IgG diluted in the proportion of 1/2000 into the covered tissue, incubating for 1h at 37 ℃, and flushing with PBST for 5 times;

the pellet was dehydrated and sealed, and the result was observed under a microscope, as shown in FIG. 12.

By combining the embodiment 2 with the comparative example and combining fig. 11 and fig. 12, it can be seen that fig. 11 has clearer background, better contrast and high staining coverage rate compared with fig. 12, which indicates that the anti-Calponin fusion nanobody disclosed in the application has higher tissue penetrability, can improve the accuracy and sensitivity of immunohistochemical detection, and is convenient for diagnosing and monitoring various diseases such as tumors in clinic.

The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.

Claims

1. An anti-calmodulin nanobody comprising a single domain heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences:

CDR-H1：LIFEIYSSA；CDR-H2：VKGR；CDR-H3：ARNPTG。

2. the anti-calmodulin nanobody of claim 1, wherein the single domain heavy chain variable region comprises the amino acid sequence shown below:

3. a high-permeability anti-calmodulin fusion nanobody, which is obtained by connecting the anti-calmodulin nanobody according to any one of claims 1 to 2 in series with horseradish peroxidase.

4. A nucleotide encoding the high permeability anti-calmodulin fusion nanobody of claim 3 and/or the anti-calmodulin nanobody of any one of claims 1-2.

5. An expression vector, characterized in that: comprising the nucleotide according to claim 4.

6. A host cell, characterized in that: expressing a high permeability anti-calmodulin fusion nanobody according to claim 3 and/or comprising a nucleotide according to claim 4 and/or comprising an expression vector according to claim 5.

7. A calmodulin detection kit comprising the high-permeability anti-calmodulin fusion nanobody of claim 3.