CN114716546A

CN114716546A - STAT3 nano antibody and application thereof

Info

Publication number: CN114716546A
Application number: CN202210317660.7A
Authority: CN
Inventors: 范瑞文; 秦蓉芬; 白祟智; 张俊珍
Original assignee: SHANXI PROVINCE CHINESE MEDICINE RESEARCH INSTITUTE; Shanxi Agricultural University
Current assignee: SHANXI PROVINCE CHINESE MEDICINE RESEARCH INSTITUTE; Shanxi Agricultural University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-07-08

Abstract

The invention relates to a STAT3 nano antibody and application thereof, belonging to the technical field of biological engineering. The amino acid sequence of the STAT3 nano antibody is shown as SEQ ID NO. 1. The STAT3 nano antibody provided by the invention can be specifically combined with STAT3 protein, can be used for detecting the expression of STAT3 in tissues by immunohistochemistry and a WesternBlotting method, and provides a method for researching the function of STAT3 gene.

Description

STAT3 nano antibody and application thereof

Technical Field

The invention relates to the technical field of biological engineering, in particular to a STAT3 nano antibody and application thereof.

Background

The signal transduction and transcription activator 3(STAT3) is a STAT family member, consists of 750-795 amino acids, has a molecular weight of about 92kDa, is phosphorylated by receptor-associated kinases to form homodimers or heterodimers, and is translocated to the nucleus. STAT3 acts as a transcriptional activator in the nucleus, playing a key role in a variety of cellular pathways, regulating cellular proliferation, differentiation, apoptosis, angiogenesis, inflammation, and immune responses. STAT3 is frequently overexpressed or deregulated in tumors, and activation of STAT3 is essential in various tumors, and its abnormal activation triggers tumor progression through expression of oncogenes, thereby promoting malignant development of tumors. Whereas activation of STAT3 in immune cells results in an increase in immunosuppressive factors. An increasing number of studies have shown that the tumor microenvironment is closely related to the STAT3 signaling pathway. Thus, targeting STAT3 may improve tumor progression, promoting an anti-tumor immune response. The STAT3 inhibitor can target STAT3 protein, inhibit phosphorylation, dimerization and nuclear translocation of the STAT3 protein in cancer cells, and enable the transcriptional function of the STAT3 protein to be lost, thereby possibly providing a new direction for disease treatment.

The antigen binding sites (VHH) of single domain antibodies in camelids (alpaca, camel) and cartilaginous fish have independent antigen recognition capabilities, also known as nanobodies. Compared with the traditional tetrad antibody, the nano antibody has the advantages of being superior to the traditional antibody in terms of small molecular weight, simple structure, stable physicochemical property and the like, and has wide prospects in the application fields of disease diagnosis, treatment and scientific research of antibodies. At present, STAT3 nano antibody has not been reported.

Disclosure of Invention

The invention aims to provide a STAT3 nano antibody and application thereof. The STAT3 nano antibody has the advantages of small inherent molecular weight, stable physicochemical characteristics, high affinity, simple structure, easy recombinant expression and preparation, capability of penetrating through a blood brain barrier and larger application prospect in diagnosis and treatment of diseases. The STAT3 nano antibody provided by the invention can be specifically combined with STAT3 protein, and can be used for detecting the expression of STAT3 in tissues by immunohistochemistry and Western Blotting method.

The invention provides a STAT3 nano antibody, wherein the amino acid sequence of the STAT3 nano antibody is shown in SEQ ID NO. 1.

Preferably, the nucleotide sequence of the gene for coding the STAT3 nano antibody is shown as SEQ ID NO. 2.

The invention also provides application of the STAT3 nano antibody in the technical scheme in preparation of an immunohistochemical reagent combined with the STAT3 protein.

The invention also provides application of the STAT3 nano antibody in the technical scheme in preparation of a Western Blotting detection reagent combined with STAT3 protein.

The invention provides a STAT3 nano antibody and application thereof. The phage display technology is utilized to carry out panning, the phage display technology can display the expressed exogenous polypeptide or protein on the surface of the phage in the form of fusion protein, and then the phage expressing specific protein is screened by an affinity enrichment method. The STAT3 nano antibody developed by adopting a phage display experimental technology can be specifically combined with STAT3 protein, and can be used for detecting the expression of STAT3 in tissues by immunohistochemistry and Western Blotting method.

Drawings

FIG. 1 is a graph showing the detection result of purified STAT3-VHH-His by Western Blot provided by the present invention;

FIG. 2 is a detection map of purified STAT3-VHH detected by Coomassie Brilliant blue provided by the invention;

FIG. 3 is a diagram showing the expression result of STAT3 protein in brain tissue detected by a STAT3-VHH nano antibody by a Western Blot method provided by the invention;

FIG. 4 is a diagram showing the result of STAT3 expression distribution in mouse brain tissue detected by STAT3 nanobody in immunohistochemistry provided by the present invention.

Detailed Description

The invention provides a STAT3 nano antibody, wherein the amino acid sequence of the STAT3 nano antibody is shown in SEQ ID NO. 1: ESGGGLVQPGGSLRLSCAASGFTFSNYAMTWVRQAPGKGPEWVSDINMAGSMTRYADSVKGRFTISRDNAKNTLYLQMDSLKPEDTGLYYCARRNVGWDGTAGEEWDFRGQGTQVTVSS are provided.

In the invention, the nucleotide sequence of the gene for coding the STAT3 nano antibody is shown as SEQ ID NO. 2: GAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGACCTGGGTCCGCCAGGCTCCAGGAAAGGGGCCCGAGTGGGTCTCAGATATTAATATGGCTGGTAGTATGACAAGGTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTACAGATGGACAGCCTGAAACCTGAAGACACGGGCCTGTATTACTGTGCGAGACGAAATGTAGGTTGGGATGGAACCGCCGGGGAAGAGTGGGACTTCCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA are provided.

In the present invention, the method for screening STAT3 nanobody preferably comprises the following steps:

1) carrying out first round of panning on the melanoma nano library to obtain B16-STAT3-VHH 1;

the coating concentration of the STAT3 protein of the first round of panning is 20 mug/ml;

2) sequentially carrying out second round, third round and fourth round elutriation on the B16-STAT3-VHH1 obtained in the step 1) to obtain a phage solution;

the coating concentration of the STAT3 protein obtained by the second round of panning is 10 mug/ml;

the coating concentration of the STAT3 protein obtained in the third round of panning is 5 mug/ml;

the coating concentration of the STAT3 protein obtained in the fourth round of panning is 5 mug/ml;

3) mixing the phage solution obtained in the step 2) with TG1 bacterial liquid, infecting and then culturing to obtain a bacterial strain;

4) mixing and infecting the strain obtained in the step 3) with KM13 helper phage, performing first shaking culture on the obtained infectious matter, performing first centrifugation, suspending the obtained first precipitate by using a liquid culture medium, performing second shaking culture, performing second centrifugation, mixing and incubating the obtained second supernatant with a confining liquid, performing indirect ELISA detection, and detecting the reactivity of the second supernatant with STAT3 protein to determine that the strain has reactivity with STAT3 protein;

the temperature of the first oscillation is 35-42 ℃, and the temperature of the second oscillation is 28-32 ℃;

the centrifugal force of the first centrifugation is 7500-8500 g, and the centrifugal force of the second centrifugation is 2000-2100 g;

5) carrying out plasmid extraction on the strain with reactivity with STAT3 protein in the step 4), carrying out PCR amplification by using a plasmid primer pair by taking the plasmid as a template to obtain a nano antibody VHH fragment, and connecting the nano antibody VHH fragment with an expression vector to obtain a recombinant plasmid;

the plasmid primers comprise a plasmid upstream primer and a plasmid downstream primer, wherein the nucleotide sequence of the plasmid upstream primer is shown as SEQ ID NO.3, and the plasmid upstream primer specifically comprises the following components:

GGGGTACCGAGTCTGGGGGAGGCTTGG；

the nucleotide sequence of the plasmid downstream primer is shown as SEQ ID No.4, and specifically comprises the following steps:

CGGGATCCTGAGGAGACGGTGACCTGG；

6) transferring the recombinant plasmid obtained in the step 5) into escherichia coli to obtain a nano antibody expression strain, carrying out IPTG induction on the nano antibody expression strain, extracting protein of the nano antibody expression strain after induction, carrying out SDS-PAGE identification and Western Blot identification on the protein, and identifying the protein as STAT3 nano antibody according to the molecular weight and his-tag label.

The melanoma nano-library is not particularly limited, and preferably is constructed by the construction method of the melanoma nano-library disclosed in Chinese patent with the application number of 201910058785.0 and the invention name of 'a construction method of a melanoma nano-antibody library'.

The melanoma nanometer library is subjected to first round of panning to obtain B16-STAT3-VHH1, and the B16-STAT3-VHH1 is subpackaged and frozen at-80 ℃.

The panning was performed using 50mM sodium carbonate/sodium bicarbonate buffer as coating buffer, at a concentration of 20. mu.g/ml, in a volume of 2ml, and the immune tubes were coated with STAT3 protein.

The elutriation method is preferably as follows:

1) mu.l melanoma Nanobody was inoculated into 100ml 2 XYTAG medium, cultured at 37 ℃ with 200rmp shaking for 1 hour to OD₆₀₀Is 0.4;

2) adding KM13 helper phage, adding 100 μ l KM13 helper phage into 100ml bacterial liquid, standing at 37 deg.C for 30min, and performing shake culture for 30 min;

3) centrifuging at 4000 Xg for 10min, removing culture medium supernatant, suspending thallus precipitate with 100ml of 2 XYTAK culture medium, and shake culturing at 30 deg.C and 200rmp overnight;

4) centrifuging overnight culture bacterial liquid at 4 ℃ for 10min at 11000 Xg in the morning the next day, transferring the supernatant to a new centrifugal bottle, adding 20ml of PEG/NaCl solution, and mixing and ice-bathing for 90 min;

5)11000 Xg, centrifuging at 4 ℃ for 30min, discarding the supernatant, then centrifuging again for 2min, and completely sucking up the supernatant;

6) resuspending the pellet with 1.3ml PBS buffer, and centrifuging at 11600 Xg for 10 min;

7) the supernatant was recovered and named STAT3-SR1, 100. mu.l of the supernatant was used for titer determination, and the remainder was mixed with 1.2ml of MPBS solution and incubated at room temperature for 1 hour to obtain a mixture (STAT 3-VHH1 treated with MPBS solution) for use.

The coating protein treatment is preferably as follows:

1) the next day after coating the proteins, the liquid in the immune tubes was decanted and the tubes were washed 3 times with PBS buffer.

2) Each tube was filled with MPBS, blocked at room temperature for 2h, and then washed 3 times with PBS buffer.

3) 2ml of the mixture obtained in the panning step 7) above was added to an immune tube, incubated at room temperature for 2 hours, and then the tube was washed 10 times with PBST solution, and then washed 10 times with PBS buffer.

4) 2ml of 100mM TEA solution was added to each tube and the bound phage eluted by gentle shaking for 15min at room temperature, followed by neutralization with 2ml of Tris-HCl solution.

5) The eluted phage (named STAT3-SC1) was transferred to a 50ml centrifuge tube, and 16ml of TG1 bacterial solution with OD600 of 0.4 was added thereto, and the solution was subjected to water bath at 37 ℃ for 30min to infect TG1 bacterial solution with the eluted phage. (and 4ml of TG1 bacteria with OD600 of 0.4 were added to the immune tube for infection and finally pooled, totaling a volume of 24 ml.)

6) 100 mul of the bacterial liquid is taken for titer determination, and the residual bacterial liquid is centrifuged for 10min at 4000 g.

7) The bacterial pellet was suspended using 1ml of 2 XYT medium, and the suspended bacterial solution was applied to 5 2 XYTAG solid plates (150mm plates) and incubated overnight at 30 ℃.

8) Collecting colonies growing on a plate by using a 2 XYT culture medium for the next day, adding 60% of glycerol to a final concentration of 15%, wherein the final concentration is a first-level library bacterium named as B16-STAT3-VHH1, and subpackaging and freezing at-80 ℃.

Determination of rescued phage titer: performing gradient dilution on STAT3-SR1, wherein the dilution is 10 < -7 > to 10 < -13 >; 10 mul of phage were used to infect 190 mul of TG1 bacterial solution with OD600 of 0.4 at each dilution; coating 100 mul of bacterial liquid of each dilution on a 2 XYTAG solid culture plate, and culturing in an incubator at 30 ℃ overnight; the colonies on the assay plate were counted and STAT3-SR1 titers were calculated.

Determination of eluted phage titer: the bacterial liquid for titer determination is diluted in a gradient from 10^-1～10^-5(ii) a Coating 100 mul of bacterial liquid of each dilution on a 2 XYTAG solid culture plate, and culturing in an incubator at 30 ℃ overnight; counting colonies on the assay plate, calculating STAT3-SC1 titer; and then calculating the input-output ratio I/O of the first round of panning.

On the basis of one round of panning, carry out two to four rounds of panning in proper order, the coating concentration of STAT3 protein of second round of panning is 10 mug/ml, the coating concentration of STAT3 protein of third round of panning is 5 mug/ml, the coating concentration of STAT3 protein of fourth round of panning is 5 mug/ml.

In the invention, the temperature of the culture in the step 3) is preferably 25-35 ℃, the infection time in the step 4) is preferably 25-35 min, and the incubation time in the step 4) is preferably 50-70 min.

In the invention, the STAT3 nano antibody has the molecular weight of 13 KD.

The STAT3 nanobody and the application thereof are further described in detail with reference to the following specific examples, and the technical solutions of the present invention include, but are not limited to, the following examples.

Example 1

The STAT3 nanobody screening method comprises the following steps:

1) performing a first round of panning on a melanoma nanockul (prepared according to a method disclosed in Chinese patent CN 201910058785.0) to obtain B16-STAT3-VHH 1;

the coating concentration of the STAT3 protein of the second round of panning is 10 mug/ml;

the coating concentration of the STAT3 protein obtained in the fourth round of panning was 5. mu.g/ml;

3) mixing the phage liquid obtained in the step 2) with TG1 bacterial liquid, infecting and culturing to obtain a strain;

the plasmid primers comprise a plasmid upstream primer (SEQ ID No.3) and a plasmid downstream primer (SEQ ID No. 4);

6) transferring the recombinant plasmid and pCold I obtained in the step 5) into escherichia coli to obtain a nano antibody expression strain, carrying out IPTG induction on the nano antibody expression strain, extracting protein of the nano antibody expression strain after induction, carrying out SDS-PAGE identification and Western Blot identification on the protein, and identifying the protein as STAT3 nano antibody according to molecular weight and his-tag label.

Performing the first round of panning from the prepared melanoma nanometer library to obtain B16-STAT3-VHH1, and subpackaging and freezing at-80 ℃.

The washing was carried out using 50mM sodium carbonate/sodium bicarbonate buffer as coating buffer, at a concentration of 20. mu.g/ml, in a volume of 2ml, and the immune tubes were coated with STAT3 protein.

The elutriation method is as follows:

1) inoculating 500. mu.l melanoma nanockulture to 100ml 2 XYTAG medium, culturing at 37 ℃ and 200rmp with shaking for 1 hour to OD₆₀₀Is 0.4;

4) centrifuging the culture solution overnight at 4 deg.C for 10min in 11000 Xg the next morning, transferring the supernatant to a new centrifugal bottle, adding 20ml PEG/NaCl solution, mixing and ice-cooling for 90 min;

6) resuspending the pellet in 1.3ml PBS buffer, then subpackaging in 2 1.5ml centrifuge tubes, and centrifuging for 10min at 11600 Xg;

Coating protein treatment:

5) The eluted phage (designated STAT3-SC1) were transferred to a 50ml centrifuge tube and 16ml OD was added₆₀₀The eluted phage was infected with TG1 bacterial solution in TG1 bacterial solution of 0.4 in 37 ℃ water bath for 30 min. (and 4ml of OD was added to the immune tube)₆₀₀0.4 TG1 inoculum was infected and finally pooled, totaling a volume of 24 ml).

6) 100 mul of the bacterial liquid is taken for titer determination, and the rest of the bacterial liquid is centrifuged at 4000g for 10 min.

Determination of rescued phage titer: STAT3-SR1 was diluted in a gradient from 10^-7～10^-13(ii) a Mu.l phage infection 190. mu.l OD was taken for each dilution₆₀₀0.4 TG1 bacterial liquid; coating 100 mul of bacterial liquid of each dilution on a 2 XYTAG solid culture plate, and culturing in an incubator at 30 ℃ overnight; the colonies on the assay plate were counted and STAT3-SR1 titers were calculated.

Determination of eluted phage titer: the bacterial liquid for titer determination is diluted in gradient from 10^-1～10^-5(ii) a Coating 100 mul of bacterial liquid of each dilution on a 2 XYTAG solid culture plate, and culturing in an incubator at 30 ℃ overnight; counting colonies on the determination plate, and calculating STAT3-SC titer; and then calculating the input-output ratio I/O of the first round of panning.

On the basis of one round of elutriation, two to four rounds of elutriations are carried out in sequence: the STAT3 protein coating concentration is 10 mu respectivelyg/ml, 5. mu.g/ml; the dilution for determining the titer of the rescued bacteriophage is respectively 10^-7～10^-12、10^-8～10^-11、10^-8～10^-11(ii) a The titer determination dilution for eluting phage M13-STAT3 was 10^-1～10^-6、10^-1～10^-6、10^-1～10^-6(ii) a After the eluted phage was neutralized with Tris-HCl solution (1M, pH 7.4), 200. mu.l of phage was infected with 800. mu.l of OD₆₀₀TG1 bacterial solution (100. mu.l of which was diluted in a gradient and the remainder was preserved) at 0.4, followed by 10^-1～10^-6Total 4 dilutions, each dilution coated with 32 × YTAG solid culture plates (150mm plates), each plate 100 μ l bacterial liquid, placed in 30 degrees C culture overnight; the plate colonies were counted, titer calculated, and the plates were labeled as plates and placed in a 4 ℃ freezer for use.

Screening of specific nano antibodies:

preparation of monoclonal phage supernatants: 96 monoclonal strains are picked from each plate and inoculated into 1 96-well deep-well culture plate, each well contains 1ml of 2 XYTAG culture medium, the culture plates are respectively marked as STAT3 library strains, and the culture plates are subjected to shaking culture at 30 ℃. After 8h, 50. mu.l of the bacterial suspension was aspirated from each well and inoculated into 500. mu.l of 2 XYTAG medium, and the mixture was subjected to shaking culture at 37 ℃ while 60. mu.l of 60% glycerol was added to the remaining bacterial suspension of the original plate to a final concentration of 15% and frozen at-80 ℃. After the plate was cultured with shaking at 37 ℃ for 1 hour, 50. mu.l of KM13 (60. mu.l of KM13+12ml of 2 XYTAG medium) helper phage was added to each well, and left to stand at 37 ℃ for infection for 30min, followed by culturing with shaking at 37 ℃ for 40 min. The deep well plate was centrifuged at 1800 Xg for 10min, the supernatant was discarded and 400ul of 2 XYTAK medium was added to each well to resuspend the pellet, followed by shaking culture at 30 ℃ overnight. The following day, centrifugation at 2020 Xg max for 20min, 250. mu.l phage supernatant from each well was pipetted into a new deep well plate and 250. mu.l blocking solution (PBS buffer containing 3% BSA) was added to each well and incubated for 1 hour at room temperature for use in indirect ELISA detection.

Identification of specific monoclonal phages: the reactivity of the phage supernatant with STAT3 protein was detected by indirect ELISA assay, as follows: design experimental group, negative control group andand the BSA control group, the experimental group and the negative control group use STAT3 protein to coat a 96-well enzyme-labeled plate, the coating concentration is 2 mug/ml, the BSA control group uses BSA protein to coat the 96-well enzyme-labeled plate, the coating concentration is 2ug/ml, each well is 100 mug, and the mixture is placed at 4 ℃ overnight. The wells were then discarded, and 100. mu.l of blocking solution was added to each well and blocked at 37 ℃ for 1 h. And (3) abandoning the blocking solution in the holes, respectively adding 100 mu l of phage supernatant obtained by four rounds of screening and treated by the blocking solution into each hole of the experimental group and the BSA control group as a primary antibody, adding the same amount of PBS into the negative control group, and incubating for 1h at 37 ℃. The plates were washed 6 times with PBST wash. Mu.l of secondary Antibody (HRP-M13 Antibody, dilution 1:6000) was added to each well and incubated at 37 ℃ for 1 h. The plates were washed 8 times with PBST wash. And adding 100 mu l of chromogenic substrate into each hole, reacting for 5-15 min in a dark place, and then adding 50 mu l of stop solution into each hole to terminate the reaction. Placing the 96-well enzyme label plate on a plate reader to read OD₄₅₀The absorption value. ELISA results were analyzed and positive strains were determined.

The glycerol corresponding to the positive well was inoculated into 5ml of 2 XYTAG medium, and after shaking culture at 37 ℃, the bacterial solution was sequenced by sequencing. And after the result of the sequence to be detected is returned, analyzing the sequencing result, selecting the strain with correct sequencing, repeating the experiment again, verifying the positive strain, and determining the recombinant plasmid constructed strain according to the ELISA identification result (shown in the table 1) of the STAT3 monoclonal positive strain.

STAT3 nanobody activity and affinity:

constructing prokaryotic expression recombinant plasmid: inoculating the glycerol strain of the clone strain with the correct sequencing result to 5ml of 2 XYTAG culture medium for culture, and extracting plasmids by using a plasmid miniprep kit to serve as template plasmids for prokaryotic expression. Then, primers for prokaryotic expression are designed, and Kpn1 and BamH1 enzyme cutting sites are respectively introduced at the 5 'end and the 3' end of the primers. The designed primer is utilized to amplify the VHH sequence of the nano antibody, and the VHH sequence is connected into a pCold I prokaryotic expression vector through the enzyme cleavage site to construct a nano antibody prokaryotic expression recombinant plasmid so as to carry out STAT3 specific identification of the nano antibody.

Primers for prokaryotic expression:

F(SEQ ID No.3):GGGGTACCGAGTCTGGGGGAGGCTTGG；

R(SEQ ID No.4):CGGGATCCTGAGGAGACGGTGACCTGG；

the screening steps are as follows:

the recombinant plasmid and pCold I were transformed into BL21(DE3) strain under no load and the corresponding nanobody expressing strain was obtained. And then carrying out induction expression on the nano antibody, wherein the specific method comprises the following steps:

and (4) carrying out overnight culture on the transformed bacteria liquid coated with the plate, and picking the monoclonal colony on the culture plate the next day for overnight culture. The bacterial liquid cultured the next day is subjected to bacteria preservation.

10 mul of glycerol bacteria are absorbed and inoculated in 5ml of Amp resistant LB culture medium, and the shaking culture is carried out overnight at 37 ℃;

the next day, 50. mu.l of the bacterial solution was aspirated and inoculated to 5ml of Amp-resistant LB, each inoculated to 2 tubes, and shake-cultured at 37 ℃ to OD₆₀₀Is 0.6;

adding IPTG into 1 tube of the bacterial liquid for induction (the final concentration is 0.4mM), and adding no IPTG into the other 1 tube of the bacterial liquid as an uninduced control for shake culture at 15 ℃ for overnight;

meanwhile, BL21(DE3) was used as an empty strain control, and an LB medium without resistance was used for the empty strain control culture.

SDS-PAGE identification of Nanobodies:

the expression of the nano antibody is identified by SDS-PAGE, and the specific method comprises the following steps:

1ml of the bacterial liquid is absorbed into a 1.5ml centrifuge tube and centrifuged for 2min at 13000 rpm;

discarding the supernatant, washing the bacterial pellet with PBS buffer solution for 2 times;

the pellet was resuspended in 20. mu.l PBS buffer, then 5. mu.l of 5 XP buffer was added and the sample was boiled in boiling water for 5 min. The samples were electrophoresed using a 10% polyacrylamide gel. After electrophoresis is finished, the gel is dyed by Coomassie brilliant blue dyeing liquid for 1h, and then, the gel is decolored by decoloration liquid.

Screening of nanobodies with neutralizing activity against STAT 3: inoculating the screened glycerol strain corresponding to the nano antibody into 5ml of Amp resistant LB culture medium, carrying out shake culture at 37 ℃ for 10h, then transferring the glycerol strain into 500ml of Amp resistant LB culture medium, carrying out shake culture at 37 ℃ until OD600 is 0.6, adding IPTG (final concentration is 0.4mM) for induction expression, and carrying out shake culture at 15 ℃ overnight. The next day, the nanobody was purified in small amounts.

Affinity of STAT3 nanobody: coating the ELISA plate with STAT3, wherein the coating concentration is 2 mug/ml and 4 mug/ml respectively; and (3) blocking the antibody with 5% of skimmed milk powder, and taking the purified STAT3 nano antibody as a primary antibody for ELISA identification.

Identification of the purified product: through ELISA identification, the STAT3 nano antibody with the best affinity is screened out, and the antibody is subjected to his label identification by a Western Blot method: after SDS-PAGE electrophoresis, the mixture is transferred to an NC membrane, and is directly marked by a his secondary antibody, and the antibody is displayed by a developing method.

As a result:

sequencing and specific monoclonal phage ELISA screening results

The phage supernatants corresponding to the monoclonals were tested for reactivity with STAT3 protein by indirect ELISA using sequencing companies to select strains that correctly express the VHH fragment clones, which were reactive to STAT3 protein to varying degrees (Table 1).

The correct and predicted amino acid sequence for sequencing is:

SEQ ID No.5(STAT3-VHH-9A)：

ESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSDINSGGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKDWGGPGTDVYDYWGQGTQVTVSS；

SEQ ID No.6(STAT3-VHH-10A)

ESGGGLVEPGGSLRLSCAASGFDFSISDMSWVREAPGKGLEWVSDIDSRGGNILYADFAKGRFTISRDNAKSTLSLQMNSLKPDDTAVYYCKDPQGLTRGQGTQVTVSS；

SEQ ID No.7(STAT3-VHH-10B)：

ESGGGLVQPGGSLRLSCAASGFTFSNYAMTWVRQAPGKGPEWVSDINMAGSMTRYADSVKGRFTISRDNAKNTLYLQMDSLKPEDTGLYYCARRNVGWDGTAGEEWDFRGQGTQVTVSS；

SEQ ID No.8(STAT3-VHH-11A)：

ESGGGLAQPGDSLRVSCVASGFTVSSHDMNWVRQAPGKGLEWVSQIDSDGSTRYADSVKGRFTISRDNAKNTLYLQMNSLNVQDTGVYYCAKGGAPSPWDWHATSLGEYDYWGQGTQVSVFS；

SEQ ID No.9(STAT3-VHH-11B)：

SEQ ID No.10(STAT3-VHH-12A)：

ESGGGLMQLGGSLRLSCAASGFTFSISPMSWVRQAPGKGLEWVSHISSHESQTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAKNRIPYYSYSYYYTGALDAWGQGTLVAVSS；

SEQ ID No.11(STAT3-VHH-12H)：

SEQ ID No.12(STAT3-VHH-6C)：

ESGGGLVQPGGSLRLSCVASRSIVVVYKMAWYRQAPGKQRELVAAISSDGKIGSSDSVKGRFTISRDDDNYSVYLQMNNLKPEDTAIYYCHAGYMAGQPGGRSYWGQGTQVTVSS；

TABLE 1 STAT3 monoclonal ELISA screening results

STAT3 nanobody affinity detection:

after ELISA detection, three clones with strong positive, namely STAT3-VHH-10A (SEQ ID No.6), STAT3-VHH-10B (SEQ ID No.7) and STAT3-VHH-12A (SEQ ID No.10), are selected as specific monoclonal positive strains for subsequent tests.

The construction of a subsequent prokaryotic expression vector is carried out on the specific monoclonal, the STAT3-VHH-10B protein expression strain is successfully constructed, and after the STAT3 nano antibody is induced, expressed and purified, the detection of a His label is carried out: carrying out His label detection on the purified antibody by using a Western Blot method, wherein FIG. 1 is a graph of the detection result of the Western Blot on the purified STAT 3-VHH-His; the purified STAT3 nano antibody is used as a Western Blot protein sample after being denatured, and HRP Anti His-Tag Mouse is used as a secondary antibody, and the result shows that the purified nano antibody is used as a protein to be detected for Western Blot identification, the purified nano antibody is detected and identified by using the HRP Anti His-Tag Mouse antibody, the molecular weight is about 13kDa after 3 experiments are repeated, the size of the nano antibody is consistent with the size of the nano antibody, and the STAT3 nano antibody is judged by the molecular weight and specific combination.

The invention takes the purified nano antibody as the protein to be detected to carry out Coomassie brilliant blue identification and judges whether the purified nano antibody is single or not. FIG. 2 is a Coomassie Brilliant blue assay map of purified STAT 3-VHH. As can be seen from fig. 2, the purified STAT3 nanobody has a single band, which indicates that nanobody purification is successful. .

FIG. 3 is a graph showing the result of detecting STAT3 protein expression in brain tissue by using STAT3-VHH nanobody in Western Blot; according to the invention, a protein sample in a Mouse brain tissue is selected as a Western Blot protein sample, purified STAT3-VHH is used as a primary antibody, HRP Anti His-Tag Mouse is used as a secondary antibody, a specific immune positive band can be detected in 3 experimental repetitions, the molecular weight is about 92Kd and is consistent with the molecular weight of STAT3 protein in related documents, and the experimental result fully indicates that the purified nano antibody can be used for the Western Blot primary antibody and has an ideal experimental effect.

FIG. 4 is a graph showing the results of detecting STAT3 expression distribution in mouse brain tissue by STAT3 nanobody in immunohistochemistry. Normal Mouse brain tissue was selected and STAT3 nanobody was used as a primary antibody and HRP Anti His-Tag Mouse was used as a secondary antibody, and the results showed that an immunopositive signal for STAT3 (B in fig. 4, D in fig. 4) was visible in the cytoplasm compared to the control group (a in fig. 4, C in fig. 4). The experimental result fully indicates that the purified nano antibody can be used for immunohistochemical detection of STAT3 localization in cells, and the experimental effect is ideal.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Shanxi university of agriculture

SHANXI TRADITIONAL CHINESE MEDICINE Research Institute

<120> STAT3 nano antibody and application thereof

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Thr Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Asp Ile Asn Met

35 40 45

Ala Gly Ser Met Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Ala Arg Arg Asn Val

85 90 95

Gly Trp Asp Gly Thr Ala Gly Glu Glu Trp Asp Phe Arg Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 2

<211> 357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gagtctgggg gaggcttggt gcaacctggg gggtctctga gactctcctg tgcagcctct 60

ggattcacct tcagtaacta tgctatgacc tgggtccgcc aggctccagg aaaggggccc 120

gagtgggtct cagatattaa tatggctggt agtatgacaa ggtatgcaga ctccgtgaag 180

ggccgattca ccatctccag agacaacgcc aagaacacgc tgtatctaca gatggacagc 240

ctgaaacctg aagacacggg cctgtattac tgtgcgagac gaaatgtagg ttgggatgga 300

accgccgggg aagagtggga cttccggggc caggggaccc aggtcaccgt ctcctca 357

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggggtaccga gtctggggga ggcttgg 27

<210> 4

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

cgggatcctg aggagacggt gacctgg 27

<210> 5

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp Ile Asn Ser

35 40 45

Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Trp Gly

85 90 95

Gly Pro Gly Thr Asp Val Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 6

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Glu Ser Gly Gly Gly Leu Val Glu Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Asp Phe Ser Ile Ser Asp Met Ser Trp Val

20 25 30

Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp Ile Asp Ser

35 40 45

Arg Gly Gly Asn Ile Leu Tyr Ala Asp Phe Ala Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Ser Thr Leu Ser Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys Lys Asp Pro Gln Gly

85 90 95

Leu Thr Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser

100 105

<210> 7

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met Thr Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Pro Glu Trp Val Ser Asp Ile Asn Met

35 40 45

Ala Gly Ser Met Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Ala Arg Arg Asn Val

85 90 95

Gly Trp Asp Gly Thr Ala Gly Glu Glu Trp Asp Phe Arg Gly Gln Gly

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210> 8

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly Asp Ser Leu Arg Val Ser

1 5 10 15

Cys Val Ala Ser Gly Phe Thr Val Ser Ser His Asp Met Asn Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Gln Ile Asp Ser

35 40 45

Asp Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

50 55 60

Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu

65 70 75 80

Asn Val Gln Asp Thr Gly Val Tyr Tyr Cys Ala Lys Gly Gly Ala Pro

85 90 95

Ser Pro Trp Asp Trp His Ala Thr Ser Leu Gly Glu Tyr Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Gln Val Ser Val Phe Ser

115 120

<210> 9

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp Ile Asn Ser

35 40 45

Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Trp Gly

85 90 95

Gly Pro Gly Thr Asp Val Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Ser Gly Gly Gly Leu Met Gln Leu Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Ser Pro Met Ser Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser His Ile Ser Ser

35 40 45

His Glu Ser Gln Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asn Arg Ile

85 90 95

Pro Tyr Tyr Ser Tyr Ser Tyr Tyr Tyr Thr Gly Ala Leu Asp Ala Trp

100 105 110

Gly Gln Gly Thr Leu Val Ala Val Ser Ser

115 120

<210> 11

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser Trp Val

20 25 30

Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Asp Ile Asn Ser

35 40 45

Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr

50 55 60

Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser

65 70 75 80

Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Trp Gly

85 90 95

Gly Pro Gly Thr Asp Val Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 12

<211> 115

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

1 5 10 15

Cys Val Ala Ser Arg Ser Ile Val Val Val Tyr Lys Met Ala Trp Tyr

20 25 30

Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Ser Ser

35 40 45

Asp Gly Lys Ile Gly Ser Ser Asp Ser Val Lys Gly Arg Phe Thr Ile

50 55 60

Ser Arg Asp Asp Asp Asn Tyr Ser Val Tyr Leu Gln Met Asn Asn Leu

65 70 75 80

Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys His Ala Gly Tyr Met Ala

85 90 95

Gly Gln Pro Gly Gly Arg Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

Claims

1. The STAT3 nanobody is characterized in that the amino acid sequence of the STAT3 nanobody is shown as SEQ ID NO. 1.

2. The STAT3 nanobody of claim 1, wherein the nucleotide sequence of the gene encoding the STAT3 nanobody is shown in SEQ ID No. 2.

3. Use of STAT3 nanobody according to claim 1 or 2 for the preparation of an immunohistochemical reagent binding to STAT3 protein.

4. Use of STAT3 nanobody according to claim 1 or 2 for the preparation of western blotting detection reagent binding to STAT3 protein.