CN111454355B - SOX6 nano antibody and application thereof - Google Patents
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Abstract
The invention provides a SOX6 nano antibody and application thereof, belonging to the technical field of biological engineering, wherein the SOX6 nano antibody has an amino acid sequence shown in SEQ ID No. 1. The SOX6 nano antibody provided by the invention can inhibit the migration of melanoma cells.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a SOX6 nano antibody and application thereof.
Background
A specific antibody in camelids (alpaca, camel) and cartilaginous fish that naturally lacks heavy chains but still is biologically active is called a single domain antibody, the antigen binding sites (VHH) of which have independent antigen recognition capabilities, and the independently expressed VHH is also called a nanobody. The nano antibody is a new generation antibody with the characteristic of small volume (nano level), has strong infiltration capacity in various tissues, has good stability in difficult environment, and is easy to produce in a microbial system. Nanobodies are actually the smallest fragments of antibodies with binding capacity. The unique properties of nanobodies make them suitable candidates for the development of new antibody-based therapeutic approaches. The structure of the nano antibody is simple and easy to express in vitro in the aspect of reducing the production cost, and meanwhile, the in vitro expression is not easy to generate inclusion bodies, so that the production process is simple; meanwhile, the nano antibody has the characteristics of small molecular weight, simple structure, more contribution to gene modification, humanized modification of the nano antibody and the like. In this regard, nanobodies are diagnostic and therapeutic applications of interest to many researchers and biopharmaceutical companies.
Melanoma is one of the most aggressive malignant cancers that occur at skin sites, formed by the carcinogenesis of melanocytes. The sex-determining region Y-box (SOX) family consists of 20 genes. These genes encode transcription factors and a High Mobility Group (HMG) cassette DNA binding domain, which is very similar to the sex-determining region (Sry) protein. The sex-determining region Y-box 6 (SOX 6) belongs to the SOXD group of most vertebrates, and also SOX5 and SOX13. The expression of SOX proteins, as transcriptional regulators, is tightly controlled by a number of signaling pathways. SOX6 is expressed in mouse melanoma cells (B16), and the down-regulation of SOX6 did not affect MITF levels in B16 cells. SOX6 inhibits Cyclin D1 gene pancreatic b-cell expression and e-globulin expression. The SOX6 can promote the growth and proliferation of melanoma cells, and the prepared SOX6 nano antibody can be a tracing tool for researching the action mechanism of the SOX6 by using a molecular imaging technology on one hand, and can be a small molecular tool for inhibiting the growth of the melanoma on the other hand.
Disclosure of Invention
In view of the above, the present invention is directed to a SOX6 nanobody and an application thereof, and the SOX6 nanobody provided by the present invention can inhibit the migration of melanoma cells.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a SOX6 nano antibody, wherein the SOX6 nano antibody has an amino acid sequence shown in SEQ ID No. 1.
The invention also provides application of the SOX6 nano antibody in the technical scheme in preparation of a medicament for inhibiting melanoma cell migration.
The invention provides a SOX6 nano antibody and application thereof, wherein the SOX6 nano antibody has an amino acid sequence shown in SEQ ID No. 1. The SOX6 nano antibody provided by the invention can inhibit the migration of melanoma cells.
Drawings
FIG. 1 SDS-PAGE and Western Blotting identification of SOX6 nano-antibody;
FIG. 2 localization of SOX6-VHH1 in B16 cells;
FIG. 3 addition of SOX6-VHH1 inhibits B16 melanoma cell proliferation migration.
Detailed Description
The invention provides a SOX6 nano antibody, wherein the SOX6 nano antibody has an amino acid sequence shown in SEQ ID No.1, and specifically comprises the following steps:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP。
in the present invention, the screening method of the SOX6 nanometer antibody preferably comprises the following steps:
1) Carrying out first round of panning on the melanoma nano antibody library to obtain B16-SOX6-VHH1;
the coating concentration of the SOX6 protein obtained by the first round of elutriation is 18-22 mug/ml;
2) Sequentially carrying out second round, third round and fourth round elutriation on the B16-SOX6-VHH1 obtained in the step 1) to obtain a phage solution;
the coating concentration of the SOX6 protein subjected to the second round of elutriation is 8-12 mu g/ml;
the coating concentration of the SOX6 protein subjected to the third round of elutriation is 3-8 mug/ml;
the coating concentration of the SOX6 protein obtained by the fourth elutriation is 3-8 mug/ml;
3) Mixing the phage liquid obtained in the step 2) with TG1 bacterial liquid, infecting and then culturing to obtain a strain;
4) Mixing and infecting the strain obtained in the step 3) with KM13 helper phage, performing first shaking culture on the obtained infected substance, 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 (enzyme-Linked immuno sorbent assay), and detecting the reactivity of the second supernatant with SOX6 protein to determine that the strain has reactivity with the SOX6 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 the SOX6 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 primer comprises a plasmid upstream primer and a plasmid downstream primer, wherein the plasmid upstream primer has a nucleotide sequence shown in SEQ ID No.2, and the specific sequence is as follows:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA wherein GGATCC is BamHI site.
The downstream primer of the plasmid has a nucleotide sequence shown in SEQ ID No. 3;
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC, where GTCGAC is Sail cleavage site.
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 to obtain protein of the induced nano antibody expression strain, carrying out Western Blotting identification on the protein according to the molecular weight of the protein and His-tag label, wherein the protein with the molecular weight of 15kDa is SOX6 nano antibody.
In the present invention, the melanoma nano antibody library is preferably a melanoma nano antibody library disclosed in chinese patent CN 201910057953.4.
In the present invention, the coating concentration of the SOX6 protein of the first round of panning is 18 to 22. Mu.g/ml, preferably 10. Mu.g/ml. The method of the first round of elutriation is not particularly limited in the present invention, and a method of conventional elutriation in the art may be used.
Sequentially carrying out second round, third round and fourth round elutriation on the obtained B16-SOX6-VHH1 to obtain a phage solution; the coating concentration of the SOX6 protein subjected to the second round of elutriation is 8-12 mu g/ml; the coating concentration of the SOX6 protein subjected to the third round of elutriation is 3-8 mug/ml; the coating concentration of the SOX6 protein obtained by the fourth panning is 3-8 mug/ml.
In the present invention, the coating concentration of the SOX6 protein of the second round of panning is 8 to 12. Mu.g/ml, preferably 10. Mu.g/ml; the coating concentration of the SOX6 protein subjected to the third round of elutriation is 3-8 mug/ml, and preferably 5 mug/ml; the coating concentration of the SOX6 protein obtained by the fourth panning is 3-8 mug/ml, preferably 5 mug/ml. The method of the second round of elutriation, the third round of elutriation and the fourth round of elutriation in the present invention is not particularly limited, and a method of conventional elutriation in the art may be used.
The obtained phage liquid and TG1 bacterial liquid are mixed, infected and cultured to obtain the bacterial strain.
In the present invention, the volume ratio of the phage liquid to the TG1 bacterial liquid is preferably 1:4. In the present invention, OD of the TG1 bacterial liquid 600 The value is preferably 0.4. In the present invention, the temperature of the culture is preferably 25 to 35 ℃, more preferably 30 ℃.
Mixing and infecting an obtained strain and KM13 auxiliary phage, performing first shaking culture on an obtained infected substance, performing first centrifugation, suspending an obtained first precipitate by using a liquid culture medium, performing second shaking culture, performing second centrifugation, mixing and incubating an obtained second supernatant with a confining liquid, performing indirect ELISA (enzyme-linked immunosorbent assay), and detecting the reactivity of the second supernatant with SOX6 protein to determine that the strain has reactivity with the SOX6 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 1700-1900 g, and the centrifugal force of the second centrifugation is 2000-2100 g.
In the present invention, the strain is preferably infected in a standing manner after being mixed with the KM13 helper phage, and the infection time is preferably 25 to 35min, preferably 30min.
In the present invention, the temperature of the first oscillation is preferably 35 to 42 ℃, more preferably 37 ℃; the temperature of the second oscillation is preferably 28-32 ℃, and more preferably 30 ℃; the centrifugal force of the first centrifugation is preferably 1700 to 1900g, and more preferably the centrifugal force of the second centrifugation is 1800g; the centrifugal force of the second centrifugation is preferably 2000 to 2100g, more preferably 2020g.
Extracting plasmids of a bacterial strain with reactivity with SOX6 protein, performing PCR amplification by using the plasmids as a template and a plasmid primer pair 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, and the plasmid upstream primer has a nucleotide sequence shown in SEQ ID No. 2; the downstream primer of the plasmid has a nucleotide sequence shown in SEQ ID No. 3.
The invention has no special limitation on the extraction of the plasmid, and can be obtained by adopting a conventional plasmid extraction method. The system and procedure used for PCR amplification in the present invention are not particularly limited, and any conventionally used system and procedure may be used. In the invention, the plasmid primers comprise a plasmid upstream primer and a plasmid downstream primer, and the plasmid upstream primer preferably has a nucleotide sequence shown in SEQ ID No.2, and specifically comprises the following components:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；
the plasmid downstream primer has a nucleotide sequence shown as SEQ ID No.3, and the specific sequence is as follows:
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC。
the method for connecting the nano antibody VHH fragment and the expression vector is not particularly limited, and the conventional connection method in the field can be adopted.
The obtained recombinant plasmid and pCold I are transferred into escherichia coli to obtain a nano antibody expression strain, IPTG induction is carried out on the nano antibody expression strain, protein of the nano antibody expression strain after induction is extracted, western Blotting identification is carried out on the protein according to the protein molecular weight (about 15 kDa) and a His-tag label, and the protein with the protein molecular weight of 15kDa is an SOX6 nano antibody.
The transferring method of the recombinant plasmid and pCold I into the escherichia coli is not specially limited, and a conventional method is adopted. The induction method for carrying out IPTG induction on the nano antibody expression strain is not specially limited except for concentration, and a conventional induction method is adopted. The method for extracting the protein of the induced nano antibody expression strain is not particularly limited, and the conventional method for extracting the protein in the microorganism is adopted. The method for the SDS-PAGE identification is not particularly limited, and the method can be performed by a conventional method.
The invention also provides application of the SOX6 nano antibody in the technical scheme in preparation of a medicament for inhibiting melanoma cell migration. The dosage form of the medicine is not particularly limited, and the SOX6 nano antibody can be prepared into a medically acceptable dosage form. In the invention, the SOX6 nano antibody is taken as the only active substance in the medicine. The content of the SOX6 nano antibody in the medicine is not particularly limited, and the conventional content of active substances in the medicine is adopted.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A melanoma nano antibody library (disclosed in Chinese patent CN 201910057953.4) reserved in a laboratory is subjected to a first round of panning to obtain B16-SOX6-VHH, and the B16-SOX6-VHH is frozen at-70 ℃.
During panning, 50mM sodium carbonate/sodium bicarbonate buffer solution is used as a coating buffer solution, the coating concentration is 20 mu g/ml, the coating volume is 2ml, and an immune tube is coated with SOX6 polypeptide protein.
The elutriation method is as follows:
(1) Inoculating 500. Mu.l melanoma nanobody library into 100ml 2 XYTAG medium, culturing at 37 deg.C and 200rmp with shaking for 1 hr to OD 600 Is 0.4;
2) Adding KM13 helper phage, adding 100 mul KM13 helper phage into 100ml bacterial liquid, standing and infecting for 30 minutes at 37 ℃, and then oscillating and culturing for 30 minutes;
3) Centrifuging at 4000 Xg for 10min, removing culture supernatant, suspending the thallus precipitate with 100ml 2 XYTAK culture medium, shaking and culturing at 30 deg.C and 200rmp overnight;
4) The next morning, 11000 Xg, centrifuging at 4 ℃ for overnight culture of bacterial liquid for 10 minutes, transferring the supernatant to a new centrifugal bottle, adding 20ml of PEG/NaCl solution, and uniformly mixing in ice bath for 90 minutes;
5) 11000 Xg, centrifuging for 30 minutes at 4 ℃, discarding the supernatant, then centrifuging for 2 minutes again, and completely sucking up the supernatant;
6) Resuspending the pellet in 2.6ml PBS buffer, then subpackaging in 2 1.5ml centrifuge tubes, and centrifuging for 10 minutes at 11600 Xg;
7) The supernatant was recovered and named ZJ-B16-SOX6-VHH1, 100. Mu.l was taken for titer determination, and the remainder was mixed with 1.6ml of MPBS solution and incubated at room temperature for 1 hour to obtain a mixed solution (SOX 6-VHH1 treated with MPBS solution) for use.
Coating protein treatment:
(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 step (7) was added to an immune tube, and after incubating at room temperature for 2 hours, the tube was washed 10 times with PBST solution, and then washed 10 times with PBS buffer.
(4) To each tube was added 2ml of 100mM TEA solution, and the bound phage was eluted by gentle shaking for 15min at room temperature, followed by neutralization with 2ml Tris-HCl solution.
(5) Eluted phage (named XT-B16-SOX6-VHH 1) were transferred to a 50ml centrifuge tube and 16ml OD was added 600 The eluted phage was allowed to infect the TG1 bacterial suspension in a 0.4 TG1 bacterial suspension in a 37 ℃ water bath for 30 minutes. (and 4ml of TG1 bacteria with OD600 of 0.4 were added to the immune tube for infection, and finally combined, total 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 10min.
(7) The pellet was suspended in 1ml of 2 XYT medium, and the suspended bacterial solution was applied to 5 2 XYT AG solid plates (150 mm plates) and incubated overnight at 30 ℃.
(8) Collecting colonies growing on the 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 which is named as B16-SOX6-VHH1, and subpackaging and freezing at-70 ℃.
Determination of rescued phage titer: the ZJ-B16-SOX6-VHH1 is diluted in a gradient of 10 -7 ～10 -13 (ii) a 10 mul of phage were used to infect 190 mul of TG1 bacterial solution with OD600 of 0.4 for each dilution; taking 100 mu l of bacterial liquid from each dilution degree, coating the bacterial liquid on a 2 XYTAG solid culture plate, and placing the bacterial liquid in an incubator at 30 ℃ for overnight culture; colonies on the assay plate were counted and the ZJ-B16-SOX6-VHH1 titer was 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 XT-B16-SOX6-VHH1 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 coating concentrations of the SOX6 polypeptide protein are respectively 10 mug/ml, 5 mug/ml and 5 mug/ml; rescued phage titer assayThe dilution is 10 respectively -7 ～10 -12 、10 -8 ～10 -11 、10 -8 ～10 -11 (ii) a The titer determination dilution for eluting phage M13-SOX6 was 10 -1 ～10 -6 、10 -3 ～10 -6 、10 -3 ～10 -6 (ii) a After the eluted phage were neutralized with Tris-HCl solution (1M, pH 7.4), 200. Mu.l of phage was infected with 800. Mu.l of OD 600 0.4 of TG1 bacterial liquid (100 mul is taken for gradient dilution, the rest is preserved), and then 10 percent is carried out -3 ～10 -6 Total 4 dilutions, each dilution coated with 32 × YTAG solid culture plates (150 mm 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: 130 monoclonal strains were picked from each plate and co-inoculated into 2 96-well deep-well plates containing 200. Mu.l of 2 XYTAG medium in each well, labeled E-1 and E-2, respectively, and subjected to shake culture at 30 ℃. After 8h, 20. Mu.l of the bacterial suspension was aspirated from each well and inoculated into 180. 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 1 hour of shaking culture in the transfer plate, 20. Mu.l of KM13 (60. Mu. lKM13+ 12ml2. Multidot. YTAG) helper phage was added to each well, and left to stand at 37 ℃ for infection for 30min, followed by shaking culture at 37 ℃ for 40min. The deep-well plate was centrifuged at 1800 Xg for 10min, the supernatant was discarded and 400. Mu.l of 2 XYTAK medium was added to each well to resuspend the pellet, followed by shaking culture at 30 ℃ overnight. The next day, the maximum rotation speed of 2020Xg centrifugation for 20 minutes, from each well sucking 250. Mu.l phage supernatant to transfer to a new deep well plate, and each well adding 250. Mu.l blocking solution (PBS buffer solution containing 3% BSA) and room temperature incubation for 1 hours, ready for indirect ELISA detection.
Identification of specific monoclonal phages: the reactivity of the phage supernatant to the SOX6 protein was detected by an indirect ELISA assay, which was carried out as follows: a96-well microplate was coated with SOX6 protein at a concentration of 2. Mu.g/ml, 100. Mu.l per well, and left overnight at 4 ℃. Abandon hole the next dayThe inner cladding liquid was added to each well 100. Mu.l of blocking liquid and blocked at 37 ℃ for 1h. Discard the blocking solution in the wells, add 100. Mu.l of phage supernatant from four rounds of screening treated with blocking solution as primary antibody in each well, incubate at 37 ℃ for 1h. The plates were washed 12 times with PBST wash. To each well was added 100. Mu.l of a secondary Antibody (HRP-M13 Antibody, dilution 1 10000) and incubated at 37 ℃ for 1h. The plates were washed 12 times with PBST wash. Add 100. Mu.l chromogenic substrate to each well, react for 5-15min in the dark, and then add 50. Mu.l stop solution to each well to terminate the reaction. Placing the 96-well enzyme label plate on a plate reader to read OD 450 The absorption value. ELISA results were analyzed and positive well numbers were determined.
The reactivity of phage supernatants corresponding to 130 monoclonals with the SOX6 protein was detected by indirect ELISA, and 20 monoclonals were selected according to the results of the indirect ELISA test, and had better reactivity with the SOX6 protein and weaker reactivity with the BSA protein. 20 single clones of the culture were sequenced by sequencing.
SOX6 nanobody expression activity and affinity:
constructing prokaryotic expression recombinant plasmid: inoculating the glycerol strain of the clone strain with the correct sequencing result to a 5ml 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 BamHI and SalI enzyme cutting sites are respectively introduced into the 5 'end and the 3' end of the primers. A designed primer is used for amplifying a VHH sequence of the nano antibody, the VHH sequence is connected into a pCold I prokaryotic expression vector through the enzymatic cleavage site, and a nano antibody prokaryotic expression recombinant plasmid is constructed to carry out SOX6 specific identification on the nano antibody.
Primers for prokaryotic expression:
(SEQ ID No.2)F:
GTGAGGATCCGAGTCTGGAGGRRGCTTGGTGCA；
(SEQ ID No.3)R:
TCTGAGTCGACTGAGGAGACGRTGACSTSGGTC。
the screening procedure was as follows
The recombinant plasmid and pCold I are transformed into BL21 (DE 3) strain in a no-load way and corresponding nano antibody expression strain is 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. And (4) preserving bacteria of the bacteria cultured the next day.
10 mul of glycerol bacteria are absorbed and inoculated in 5ml of Amp resistant LB culture medium, and shake culture is carried out at 30 ℃ for overnight;
the next day, 50. Mu.l of the bacterial solution was aspirated and inoculated with 5ml of Amp-resistant LB medium, each inoculated with 2 tubes, and shake-cultured at 37 ℃ to OD 600 Is 0.6;
adding IPTG into 1 tube of the bacterial liquid for induction (the final concentration is 0.2 mM), 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 (DE 3) is used as an empty strain control, and an LB culture medium without resistance is used for the empty strain control culture.
And (3) SOX6 nano antibody purification:
subpackaging 1L SOX6 nano antibody bacterial liquid successfully expressed by induction, centrifuging at 11000 Xg for 15min at 4 ℃, and discarding the supernatant. The bacterial pellets were resuspended in 100ml PBS and then dispensed into 50ml centrifuge tubes, each containing 25ml. And (3) respectively carrying out ultrasonic crushing treatment on the bacterial liquid in a 50ml centrifugal tube in an ultrasonic crusher. After sonication was complete, the cells were centrifuged at 16500 Xg for 10min, and the sonicated supernatant was collected and filtered through a 0.02 μm filter for purification on machine (AKTApure).
According to the size of the purified protein, 0.5MPa of pre-column pressure and 2MPa of system pressure are set. After all the SOX6 nanometer antibody ultrasonic samples are loaded on the column, 5%,10%,20%,30%,40%,50% and 60% imidazole (the concentration of imidazole is 500 mM) is respectively set for elution. And recovering the eluent to identify whether the target protein is present.
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 sucked 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 minutes. 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.
Affinity of SOX6 nanobody:
coating the ELISA plate with 5. Mu.g/ml SOX 6; after BSA blocking, the purified and diluted SOX6 nano antibody is used as a primary antibody, and is respectively diluted to 5 mu g/ml, 2.5 mu g/ml, 1.25 mu g/ml, 0.625 mu g/ml and 0.3125 mu g/ml in a gradient manner for ELISA identification.
As a result:
ELISA screening results
The reactivity of 130 monoclonal phage supernatants with SOX6 protein was tested by indirect ELISA, and 10 monoclonal antibodies were selected according to the results of the indirect ELISA test, which were both more reactive with SOX6 protein and less reactive with BSA protein (Table 1). 10 single clones of the culture were sequenced by sequencing.
TABLE 1SOX6 monoclonal ELISA screening results
After ELISA screening, two clones with the strongest positive are SOX6-VHH1 and SOX6-VHH2, all 10 clones are sequenced, and the amino acid sequences after sequencing and prediction are as follows:
n.1. (SEQ ID No. 1) (SOX 6-VHH 1) amino acid sequence:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVSSAHHSEDPSSRPLWP；
n.2. (SEQ ID No. 4) (SOX 6-VHH 2) amino acid sequence:
ESGGGSVQPGGSLRLSCAASGFTFSGYYMSWVRQAPGEEPEWVTFITNDGSGVRYADSVKGRFTVSRNNVENTVYLRMDNLQPNDTARYYCVRGRLTATSPLIPDDSWGQGTQVTVSS；
n.3 (SEQ ID No. 5) (SOX 6-VHH 3) amino acid sequence:
ESGGGLVQPGGSLRLSCLASGFSFDSYAMSWYRQAPGKEREWVAHITSGGSTNYSDSVKGRFTISRDNAKNAVYLQMDNLKPEDTAVYYCNEVSTSLDDYDYWGKGTQVTVSA；
n.4 (SEQ ID No. 6) (SOX 6-VHH 4) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVS；
n.5. (SEQ ID No. 7) (SOX 6-VHH 5) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；
n.6 (SEQ ID No. 8) (SOX 6-VHH 6) amino acid sequence:
ESGGGLVQPGGSLRLSCAASGFTLGGWNIGWFRQAPGKEREGVLCISDSGESVYYLDSVKGRFTISSDYAENTVYLQMNSLKPEDTAIYFCAATYYRCSDYAPEFSSWGQGTQVTVS；
n.7 (SEQ ID No. 9) (SOX 6-VHH 7) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFTLSSYNMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；
n.8 (SEQ ID No. 10) (SOX 6-VHH 8) amino acid sequence:
ESGGGLVQPGGSLRLSCLAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYDYWGQGIQVTVSS；
n.9 (SEQ ID No. 11) (SOX 6-VHH 9) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMNTLKPEDTALYYCAKRNRAGLSAYSSWGQGTQVTVSS；
n.10. (SEQ ID No. 12) (SOX 6-VHH 10) amino acid sequence:
ESGGGLVQPGGSLRLSCAAPGFSLSSYQMSWVRQSPGKGPEWVSTIAASSGNTWYADSVKGRFTISKDNAKNTLYLQMDSLKPEDTALYYCAKRNRAGLDAYDYWGQGIQVTVSS。
detecting the affinity of the SOX6 nano antibody:
after the two strongest positive clones are selected for expression, the affinity detection result is as follows: the sensitivity of the SOX6 nano antibody N.1 to the SOX6 protein is 1.25 mu g/ml; the sensitivity of SOX6 nanobody N.2 to SOX6 protein was 2.5. Mu.g/ml.
As can be seen from the above examples, the SOX6 nanobody provided by the present invention can specifically bind to SOX6 protein, and the sensitivity to the protein is 1.25. Mu.g/ml.
3. Expression and purification of SOX6 Nanobody 1
The SOX6 nano antibody is successfully eluted at the concentration of 40% imidazole, and a single band with the size of 15kDa is identified by SDS-PAGE. Western Blotting identification is carried out on the protein according to the molecular weight of the protein and a His-tag label, and the protein with the molecular weight of 15kDa is a SOX6 nano antibody. The experimental results are shown in FIG. 1.
Example 2
Cell culture and SOX6 nanobody function: SOX6 was found to be highly expressed in melanoma cells and to be important in cell proliferation and migration. SOX6 nanobodies were identified by cell scratch experiments and cell immunohistochemistry. Culturing B16 cells in DMEM high-glucose medium containing 10% FBS, digesting and passaging when the cells grow to 80% in 6-well plate, transferring a portion of the cells 1:2 to a new 6-well plate and another portion to a non-toxic slide glass, shaking, standing at 37 deg.C for 5% CO 2 The culture was carried out overnight in an incubator.
Chemical localization of immune cells: after the B16 cells of the glass slide are full, washing the glass slide for 3 times by PBS, and then adding 500 mu l of 4% paraformaldehyde to stand for 30min at the temperature of 4 ℃; after that, wash 3 times with PBS and add 3% H 2 O 2 Standing the solution for 15min; fully washing with PBS, adding bovine serum, sealing for 20min, adding SOX6 nanometer antibody, and incubating at 4 deg.C overnight; the incubation box is placed at room temperature for 30min again on the next day, and then washed by PBS for 3 times; the secondary antibody with His tag was added and incubated at 37 ℃ for 30min, washed 3 times with PBS, and 50. Mu.l of DAB was added dropwise and incubated in a cassette for 3-10min before observation under a microscope. And (3) dripping distilled water to stop dyeing after the color development is finished, adding hematoxylin for nuclear dyeing after the distilled water is removed, and flushing with tap water to return blue after 30 s.
Cell scratch experiment identification: after B16 cells cultured by a DMEM high-sugar culture medium grow into a layer in a 6-pore plate, 1 straight line is drawn in the culture hole by using a disinfected ruler and a middle gun head, and the gun heads are kept vertical during drawing. PBS is washed for 3 times, dropped cells are removed, and serum-free DMEM high-sugar medium is added again and placed in an incubator for culture. The same location was recorded every 6 hours and photographed.
As a result:
localization of SOX6 Nanobodies in B16 cells
The control group was supplemented with an equal amount of PBS and the experimental group was supplemented with SOX6 nm antibody at a concentration of 100. Mu.g/ml. According to the results, the following results are found: the SOX6 nano-antibody is mainly distributed in the cytoplasm of the B16 cell, and the nano-antibody can enter the cell to play a role. The results of the experiment are shown in FIG. 2 (arrows).
2. Influence of addition of SOX6 nano antibody on B16 cell scratch experiment
The control group was supplemented with an equal amount of PBS and the experimental group was supplemented with SOX6 nm antibody at a concentration of 100. Mu.g/ml. According to the results, the following results are found: the group to which PBS was added was scratched to grow substantially full of cells within 36 hours. The experimental group is most obvious at 24 hours, about 20% of the experimental group is not overgrown at 36 hours, and the results show that the SOX6 nano antibody plays a role in inhibiting the migration of the B16 cells along with the increase of time after being added into the B16 cells. The results of the experiment are shown in FIG. 3.
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
<120> SOX6 nano antibody and application thereof
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 132
<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 Leu Gly Gly Trp Asn Ile Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Leu Cys Ile Ser Asp
35 40 45
Ser Gly Glu Ser Val Tyr Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Ser Asp Tyr Ala Glu Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Ile Tyr Phe Cys Ala Ala Thr Tyr Tyr
85 90 95
Arg Cys Ser Asp Tyr Ala Pro Glu Phe Ser Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala His His Ser Glu Asp Pro Ser Ser Arg
115 120 125
Pro Leu Trp Pro
130
<210> 2
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtgaggatcc gagtctggag grrgcttggt gca 33
<210> 3
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tctgagtcga ctgaggagac grtgacstsg gtc 33
<210> 4
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr Tyr Met Ser Trp Val
20 25 30
Arg Gln Ala Pro Gly Glu Glu Pro Glu Trp Val Thr Phe Ile Thr Asn
35 40 45
Asp Gly Ser Gly Val Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Val Ser Arg Asn Asn Val Glu Asn Thr Val Tyr Leu Arg Met Asp Asn
65 70 75 80
Leu Gln Pro Asn Asp Thr Ala Arg Tyr Tyr Cys Val Arg Gly Arg Leu
85 90 95
Thr Ala Thr Ser Pro Leu Ile Pro Asp Asp Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 5
<211> 113
<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 Leu Ala Ser Gly Phe Ser Phe Asp Ser Tyr Ala Met Ser Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Ala His Ile Thr Ser
35 40 45
Gly Gly Ser Thr Asn Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Arg Asp Asn Ala Lys Asn Ala Val Tyr Leu Gln Met Asp Asn Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Glu Val Ser Thr Ser
85 90 95
Leu Asp Asp Tyr Asp Tyr Trp Gly Lys Gly Thr Gln Val Thr Val Ser
100 105 110
Ala
<210> 6
<211> 114
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Ser Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110
Val Ser
<210> 7
<211> 115
<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 Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Ser Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 8
<211> 117
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
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 Leu Gly Gly Trp Asn Ile Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Leu Cys Ile Ser Asp
35 40 45
Ser Gly Glu Ser Val Tyr Tyr Leu Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Ser Asp Tyr Ala Glu Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Ile Tyr Phe Cys Ala Ala Thr Tyr Tyr
85 90 95
Arg Cys Ser Asp Tyr Ala Pro Glu Phe Ser Ser Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser
115
<210> 9
<211> 115
<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 Pro Gly Phe Thr Leu Ser Ser Tyr Asn Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Ser Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 10
<211> 115
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Leu Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Ser Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 11
<211> 115
<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 Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Ser Ala Tyr Ser Ser Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
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 Ala Ala Pro Gly Phe Ser Leu Ser Ser Tyr Gln Met Ser Trp Val
20 25 30
Arg Gln Ser Pro Gly Lys Gly Pro Glu Trp Val Ser Thr Ile Ala Ala
35 40 45
Ser Ser Gly Asn Thr Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asp Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Lys Arg Asn Arg
85 90 95
Ala Gly Leu Asp Ala Tyr Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr
100 105 110
Val Ser Ser
115
Claims (2)
1. An SOX6 nano antibody, which is characterized in that the amino acid sequence of the SOX6 nano antibody is shown as SEQ ID No. 1.
2. Use of the SOX6 nanobody of claim 1 in the preparation of a medicament for inhibiting the migration of melanoma cells.
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