CN117736322B

CN117736322B - Anti SYCE2 single-molecule antibody T2D and application thereof

Info

Publication number: CN117736322B
Application number: CN202410190959.XA
Authority: CN
Inventors: 王旻; 赵泽云; 李韬; 赖炜炜; 郭宇; 梁峰; 杨扬; 齐芮; 郭子健; 昌龙; 张博翔; 赵洪宇
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2024-02-21
Filing date: 2024-02-21
Publication date: 2024-04-19
Anticipated expiration: 2044-02-21
Also published as: CN117736322A

Abstract

The invention discloses an anti-SYCE 2 single-molecule antibody T2D and application thereof, belonging to the technical field of molecular biology, and the inventor discovers that SYCE protein is highly expressed in colon cancer tissues and is lower in normal colon tissues through a plurality of years of researches, and the inventor screens a plurality of complete anti-SYCE single-molecule antibodies in Tomlinson I+J phage scFv. Wherein, the anti SYCE2 single-molecule antibody T2D has strong specificity and high sensitivity; can be used for diagnosing colon cancer.

Description

Anti SYCE2 single-molecule antibody T2D and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-SYCE 2 single-molecule antibody T2D and application thereof in colon cancer detection.

Background

SYCE 2A protein coding gene is located at 13.13 of 19p chromosome in nucleus. Related pathways include meiosis and cell cycle, and mitosis. SYCE2 tetramers interact directly with TEX2 dimers through the frizzled domain of SYCE 12. James m. Dunce et al found that SYCE and TEX12 proteins formed a fibrous complex by interaction through techniques such as X-ray crystallography and electron microscopy. These fibers gradually extend during the chromosome association process, facilitating pairing of homologous chromosomes. In addition, there is a unique tandem structure between SYCE domains that plays a critical supporting and regulatory role in the interaction of SYCE and TEX 12. SYCE2 expression is regulated by a demethylation-dependent process. SYCE2 and TEX12 form a highly stable constitutive complex, and biophysical analysis showed that the SYCE 2-TEX 12 complex is an equimolar iso-octamer, formed by combining a SYCE2 tetramer and two TEX12 dimers.

Disclosure of Invention

The invention aims to provide an anti-SYCE 2 single-molecule antibody T2D with high sensitivity and application thereof in preparing a kit for diagnosing colon cancer.

The base sequence of the anti-SYCE 2 single-molecule antibody T2D is shown in a sequence table SEQ ID NO. 3.

Application of anti-SYCE 2 single-molecule antibody T2D shown in a sequence table SEQ ID NO.3 in preparation of kit for detecting colon cancer;

the colon cancer is colon cancer SW48 cells and colon cancer SW480 cells;

the kit is an immunohistochemical kit.

The invention provides an anti-SYCE 2 single-molecule antibody T2D and application thereof, the inventor discovers that SYCE protein is highly expressed in colon cancer tissues and is not expressed in normal colon tissues through a plurality of years of researches, and the inventor screens a plurality of complete anti-SYCE single-molecule antibodies in Tomlinson I+J phage scFv. Wherein, the anti SYCE2 single-molecule antibody T2D has strong specificity and high sensitivity; can be used for diagnosing colon cancer.

Drawings

FIG. 1 SYCE2 is a diagram of agarose gel electrophoresis analysis of PCR products;

FIG. 2 PCR identification of anti-SYCE positive phage antibody strain;

FIG. 3 results of identification of anti-SYCE 2 single-molecule antibody T2D protein after purification;

FIG. 4 shows the result of a western blot of a commercially available polyclonal antibody;

FIG. 5 shows the results of a T2D western blot of anti-SYCE 2 single-molecule antibodies;

FIG. 6 SYCE2 high expression tissue sections of anti SYCE2 single molecule antibody immunohistochemical assay;

FIG. 7 SYCE2 low expression tissue sections of anti SYCE2 single molecule antibody immunohistochemical experiments.

Detailed Description

EXAMPLE 1 construction, expression of SYCE2 protein recombinant expression plasmid pET-SUMO-SYCE2 and purification of SYCE protein

Primers P1, P2 of SYCE2 were designed and synthesized:

P1：5’-GAATTCATGGAGCGACAGGGAGTGGAC-3’；

P2: 5’-TTCGAATCAGCATTCACCATCTCTGTTG-3’；

The cDNA of a colon cancer cell line SW48 is used as a template for PCR amplification SYCE protein genes (the base sequence is shown as a sequence table SEQ ID NO. 1), and cloned into a pET-SUMO vector to construct plasmid pET-SUMO-SYCE, and then transferred into T-shot competent cells, and an agar plate containing kanamycin resistance is subjected to primary screening. Selecting single colony to culture in LB liquid culture medium; extracting plasmid by using a plasmid recovery kit and carrying out PCR identification, analyzing the product by using 1% agarose gel electrophoresis to obtain a band of about 654 bp, the size of the band is consistent with that of an inserted target gene (figure 1), and carrying out sequencing verification to prove that the target fragment is correctly inserted into a vector, thereby successfully constructing a recombinant plasmid pET-SUMO-SYCE;

After the recombinant plasmid pET-SUMO-SYCE is transformed into an escherichia coli BL21 strain, single colonies are respectively picked up and inoculated into 5ml of LB culture medium containing ampicillin, and shake culture is carried out at 37 ℃ for overnight; then the bacterial liquid is mixed according to the proportion of 1:50 in a fresh 100mL culture medium, continuously culturing until the OD value reaches about 0.8, adding IPTG with the final concentration of 1mmol/L, continuously culturing at 37 ℃ for 6 hours. Because the pET-SUMO carrier contains the His tag, the recombinant SYCE protein is obtained according to the conventional operation steps of recombinant His tag protein.

EXAMPLE 2 phage Single-chain antibody library

The Tomlinson I+J phage scFv was inoculated into 200 mL of 2×TY medium, 100. Mu.g/mL of Amp and 1% glucose were added, and the culture was performed at 37℃with shaking until the OD600 was about 0.4. 50mL bacterial liquid was taken out from the culture liquid, 2X 10 ¹¹ helper phage KM13 was added, and the mixture was left in a water bath at 37℃for 30 min. The bacterial solution was centrifuged at 20 min at 4℃and 3300g, and the pellet was resuspended in 100 mL of 2 XSY medium (containing 100. Mu.g/mL Amp, 50. Mu.g/mL Kan and 0.1% glucose) and incubated overnight at 250rpm with shaking at 30 ℃. The bacterial liquid cultivated overnight is centrifugated at 4 ℃,3300 Xg for 30min, 20 mL ice-cold PEG/NaCl solution (the final concentration is 20 percent PEG-6000,2.5mol/L NaCl) is added into the supernatant 80 mL, the mixture is placed on ice for more than 1h, the PEG/NaCl solution is discarded by the centrifugated at 3500 Xg for 30min at 4 ℃, the sediment is resuspended by 2mL PBS, 11600 Xg, the temperature is 4 ℃, the centrifugation is 10min, the supernatant is transferred into a sterile centrifuge tube, and the mixture is preserved at 4 ℃ (or 15 percent glycerol with the final concentration is added and preserved at-70 ℃); phage titer assays were performed simultaneously for antibody library screening.

Example 3 screening of anti-SYCE 2-scFv

The purified SYCE protein is taken as antigen to be coated on a 96-well ELISA plate, and each well is coated with 100 mu L of the antigen at 4 ℃ overnight. The next day the supernatant was discarded, the 96-well ELISA plate was washed 3 times with PBS, the unadsorbed antigen in the plate was washed off, and blocked with 3% BSA-PBS for 2h at 37 ℃. The blocking solution was discarded and the 96-well elisa plate was washed 3 times with PBS. Phage antibodies prepared in example 2 (titer 1.0X10 ¹²~1.0×10¹³) were suspended in 3% BSA-PBS and added to 96-well ELISA plates, incubated at room temperature for 60min, and unbound phage antibodies in the wells were discarded. In the first round of screening, the detergent was removed by washing 10 times with PBS containing 0.1% Tween-20 and 10 times with PBS. In the second and subsequent rounds of screening, PBS was used for washing 20 times, and non-specifically adsorbed phage was washed thoroughly. Finally, washing with distilled water for 2 times, and after washing, lightly beating the liquid remained in each hole, adding 100 mu L eluent (1 mg/mL pancreatin-PBS) into each hole for room temperature digestion for 30min, and eluting phage. 1.75mL E.Coli TG1 with OD600.apprxeq.0.4 was added to 250. Mu.L of eluted phage and incubated at 37℃for 30min without shaking. mu.L of 10 ^-4、10^-6 -fold dilution was pipetted onto a TYE plate (containing 100. Mu.g/mL Amp+1% glucose). Incubated overnight at 37℃for determination of phage titer. The remaining E.Coli TG1 medium was centrifuged at 11600g for 5min at 4℃and the pellet was resuspended in 50. Mu.L of 2 XTY medium and plated on TY plates (containing 100. Mu.g/mL amp+1% glucose) and incubated overnight at 37 ℃.

The following day, 2mL of 2 XTY medium was added to TY plates full of bacterial clones, and the bacteria were scraped with a glass coating rod, the bacterial suspension was collected, 50. Mu.L was added to 50mL of 2 XTY medium, 100. Mu.g/mL of Amp, 1% glucose was added to the medium, and shake culture was performed at 37℃until OD600 was approximately 0.4. Adding 15% glycerol into the rest bacterial liquid, and preserving at-70deg.C for use. 10mL of the culture was added to 5X 10 ¹⁰ helper KM13 at a ratio of bacteria count/helper count=1/20, and the mixture was placed in a water bath at 37℃for 30 minutes. The bacterial liquid was centrifuged at 3000g for 10min at 4℃and the resuspended bacterial cells were precipitated in 50mL of 2 XSY medium (containing 100. Mu.g/mL amp+50. Mu.g/mL Kan+0.1% glucose) and cultured overnight at 30℃with shaking. The overnight culture was centrifuged at 3300 Xg for 30min at 4℃and the supernatant was collected, added with 1/5 volume of PEG/NaCl, thoroughly mixed and ice-bathed for 60min. Centrifuge at 3300 Xg for 30min at 4℃and discard the supernatant thoroughly. The pellet was resuspended in 2mL PBS and centrifuged at 11600 Xg at 4℃for 10min, and the residual bacterial debris was removed while phage titer was determined. The obtained supernatant is the phage antibody of the first round of enrichment affinity screening. The enrichment affinity screening process was repeated 4 times, and 4 rounds of enrichment affinity screening process were performed.

50 Mu L of infection E.Coli HB2151 is taken after dilution of phage antibody 10 ^-4、10^-6 subjected to enrichment screening of round 4, after induced expression, ELISA is used for identification, and an enzyme-labeled spectrophotometer is used for measuring OD value (wavelength 450 nm). OD values were adjusted to zero with 2% Milk-PBS from control wells, double well assays were performed on each specimen and OD averages were taken. Standard negative controls were set to determine positive clone strains. Positive clone strain determination criteria were: OD value is 3 times or more than that of negative control value.

Synthesizing a universal PCR primer to amplify the ScFv whole gene fragment according to the gene sequence of pIT-2 vectors on the Tomlinson I+J kit;

LMB3: 5’—CAGGAAACAGCTATGAC—3’；

pHEN: 5’ —CTATGCGGCCCCATTCA—3’。

the fragment (figure 4) with about 756bp is amplified, the base sequence of the anti-SYCE 2 single-molecule antibody T2D is shown in a sequence table SEQ ID NO.3, and the amino acid sequence is shown in a sequence table SEQ ID NO. 4.

EXAMPLE 4 expression and purification of SYCE2-ScFv

ELISA positive strains were transferred to 500mL of 2 XTY medium (containing 100. Mu.g/mL amp+0.1% glucose) and incubated overnight at 37 ℃. The following day, 200. Mu.L of overnight cultures were transferred to 2 XTY medium (containing 100. Mu.g/mL ampicillin and 0.1% glucose) for induction of scFv expression (1.0 mmol/L IPTG, induction 16 h at 30 ℃). The supernatant was collected by centrifugation, and the target protein was precipitated with 40% saturated ammonium sulfate, diluted with 20 mL equilibrium solution (25 mmol/L Tris,0.5 mol/L NaCl, pH 7.5), and then a crude extract was prepared. The HisTrap affinity column was equilibrated with the equilibration solution. Loading at 0.5 mL/min, and eluting with equilibrium solution to reach baseline. Finally, the target protein was eluted with a gradient of eluent (25 mmol/L Tris,0.5 mol/L NaCl,200 mmol/L imidazole, pH 7.5) and the target protein peak was collected, the eluted sample was dialyzed overnight against PBS, and 10% SDS-PAGE analysis showed the target protein to be approximately 27000 Da (FIG. 3).

Example 5: detection experiment of anti SYCE2 single molecule antibody T2D

1. Extraction of Total protein of colon cancer cell line SW48 and SW480

1. Resuscitation of colon cancer cell line SW48 and SW480

1) Taking out the frozen tube containing the cells, rapidly placing the frozen tube into a water bath kettle at 37 ℃ to enable the frozen tube to be rapidly melted for 2-3min, and rapidly transferring the frozen tube to an ultra-clean bench.

2) The lid was opened, the cell suspension was aspirated into the centrifuge tube, and 2 times the amount of complete medium of the cell suspension was added and mixed (complete medium was prepared from basal medium and serum according to 9:1 ratio mixing).

3) Centrifuge at 1000rpm/min for 5min.

4) The supernatant was discarded, 1mL of complete medium was added to resuspend the cells, and the cells were gently mixed with gentle stirring to disperse the cells evenly.

5) 1ML of the cell suspension was aspirated by a pipette tip, slowly dropped into a 10cm dish containing 7mL of complete medium, shaken well, and placed in an incubator at 37℃with 5% CO ₂ for cultivation.

6) The following day under a microscope, the cell morphology and growth conditions were observed, and liquid exchange or passaging was performed according to the circumstances.

2. Enrichment of cancer cell antigens

1) When the cells in the culture dish grow to 80-90%, the culture medium in the culture dish is sucked off, 1mL precooled PBS buffer solution is added for cleaning 3 times, and the 3 rd PBS is not sucked off.

2) The cells were scraped using a cell brush, ensuring complete scraping of the membrane formed by the adherent cells, and transferred to a 1.5mL EP tube using a pipette.

3) Centrifuging at 3000rpm for 5min, removing supernatant, adding 500uL of prepared RIPA lysate at the bottom, blowing and mixing with gun head, and performing ice lysis at 20 min.

4) Centrifuging at 12,000 rpm for 30min at 4deg.C, collecting supernatant, and placing into new EP tube to obtain total cell protein.

2. And (5) performing a western blot experiment.

A rapid configuration kit of SDS-PAGE color gel produced by SparkJade company is used, total protein of colon cancer cell lines SW48 and SW480 is taken as an antigen, a monoclonal antibody T2D of SYCE2 and a commercial polyclonal antibody are taken as primary antibodies, and a western blot experiment is carried out, and the results are shown in figures 4 and 5.

3. Immune agar amplification test

The agar diffusion test was performed with the total protein of colon cancer cell line SW48 as antigen, anti-SYCE 2 single-molecule antibody T2D, and with the commercially available polyclonal antibody as control. The results are shown in Table 1

4. Selecting colon cancer tissue specimens stored in a second hospital of Jilin university, and performing immunohistochemical experiments

1. Taking out the tissue chip, and baking the chip at 70deg.C for 70min;

2. dewaxing with xylene solution, soaking for 20min, soaking with gradient alcohol for 20min, and washing with distilled water;

3. Heating in boiling sodium citrate antigen retrieval liquid for 2.5min, cooling for 10min to normal temperature, and washing with PBS for 3 times, each time for 5min;

4. Adding endogenous peroxidase blocker, blocking for 10min, and washing with PBS for 3 times each for 5min;

5. Dripping goat serum sealing liquid, and sealing for 15min;

6. Dripping anti SYCE2 single molecule antibody T2D as primary antibody, diluting at a ratio of 1:50, and incubating at 4deg.C overnight;

7. placing the mixture at normal temperature for 1h, and flushing the primary antibody by PBS;

8. Dripping secondary antibody, incubating for 20min at room temperature, and washing 3 times with PBS;

9. preparing DAB color development liquid, soaking in clear water after finishing color development, and flushing with running water;

10. After 3min of hematoxylin dip dyeing, washing with running water;

11. soaking in gradient alcohol for 2min, blow drying, sealing with neutral resin, and observing under microscope (see fig. 6 and 7).

Claims

1. An anti-SYCE 2 single-molecule antibody T2D, characterized in that: the base sequence of the polypeptide is shown in a sequence table SEQ ID NO. 3.

2. The application of the anti-SYCE 2 single-molecule antibody T2D with the base sequence shown in a sequence table SEQ ID NO.3 in preparing a kit for diagnosing colon cancer.

3. The use according to claim 2, characterized in that: the kit is an immunohistochemical kit.