Monoclonal antibody of cytokeratin component 19D as well as preparation method and application thereof

Abstract

The invention discloses a monoclonal antibody G2 of a cytokeratin component 19D as well as a preparation method and application of the monoclonal antibody G2, and relates to the technical field of tumor immunology and biological medicine, the monoclonal antibody G2 of the cytokeratin component 19D is specifically bound with the protein of the cytokeratin component 19D; the monoclonal antibody G2 of the cytokeratin component 19D comprises a heavy chain variable region and a light chain variable region, a phage display technology is utilized, screening is carried out in a human phage display antibody library Tomlinson I + J containing an artificially synthesized sequence, and a strain of monoclonal antibody is successfully separated. The amino acid sequences of CDR1, CDR2 and CDR3 regions of the heavy chain and the light chain of the monoclonal antibody are novel and can be specifically combined with the cytokeratin component 19D, and a fluorescence quenching body detection technology is developed based on the antibody and is used for simple, convenient, rapid and high-sensitivity detection of the lung cancer marker cytokeratin component 19D.

Description

Technical Field

[0001] The present invention relates to the fields of tumor immunology and biomedical technology, and specifically relates to a monoclonal antibody G2 against cytokeratin component 19D, its preparation method and application. Background Art

[0002] Cytokeratin component 19D, also known as cytokeratin 19 fragment, is a soluble acidic protein belonging to the cytokeratin family. It is mainly in the cytoplasm of monolayer and stratified epithelial tumor cells and is composed of soluble fragments of CK19-type keratin. In the field of clinical applications, cytokeratin component 19D has a wide range of uses and can be used as a non-organ-specific tumor marker, and its utility is particularly prominent in the detection of lung cancer. For non-small cell lung cancer, the detection rate can reach 70-85%. During the tumor development process, once tumor cells show lysis and death, a large number of proteases are immediately activated, resulting in the degradation of CK19-type keratin, and the generated cytokeratin component 19D is released into the blood circulation system, causing a significant increase in the level of this component in the serum. Therefore, the content of serum cytokeratin component 19D can accurately reflect the occurrence and development trend of tumors. The level of cytokeratin component 19D is positively correlated with the clinical stage of non-small cell lung cancer. After radical resection of lung cancer patients, the concentration of this component will decrease significantly. If its concentration increases again, it is very likely to indicate tumor recurrence or disease progression. Therefore, accurately monitoring the concentration level of serum cytokeratin component 19D is of great significance.

[0003] Therefore, there is an urgent need to develop an antibody that can accurately monitor the concentration level of serum cytokeratin component 19D, which can bind to cytokeratin component 19D with high affinity and high specificity, so as to accurately determine the concentration of cytokeratin component 19D in tumors. Summary of the Invention

[0004] To solve the above problems, the purpose of the present invention is to provide a monoclonal antibody G2 against cytokeratin component 19D, its preparation method and application.

[0005] To achieve the above object, the present invention is realized through the following technical solutions:

[0006] A monoclonal antibody G2 against cytokeratin component 19D, wherein the monoclonal antibody G2 against cytokeratin component 19D specifically binds to cytokeratin component 19D protein; the monoclonal antibody G2 against cytokeratin component 19D includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes three complementarity-determining regions, namely CDRH1, CDRH2 and CDRH3; the light chain variable region includes three complementarity-determining regions, namely CDRL1, CDRL2 and CDRL3;

[0007] The amino acid sequence of the heavy chain variable region of the monoclonal antibody G2 against cytokeratin component 19D is SEQ ID NO: 1, the amino acid sequence of CDRH1 is SEQ ID NO: 2, the amino acid sequence of CDRH2 is SEQ ID NO: 3, and the amino acid sequence of CDRH3 is SEQ ID NO: 4;

[0008] The amino acid sequence of the light chain variable region of the monoclonal antibody G2 against cytokeratin component 19D is SEQ ID NO: 5, the amino acid sequence of CDRL1 is SEQ ID NO: 6, the amino acid sequence of CDRL2 is SEQ ID NO: 7, and the amino acid sequence of CDRL3 is SEQ ID NO: 8.

[0009] The present invention also includes a method for preparing the monoclonal antibody G2 against cytokeratin component 19D, comprising the following steps:

[0010] ① Amplification of the Tomlinson I+J phage display antibody library;

[0011] ② Panning of the Tomlinson I+J phage display antibody library;

[0012] ③ Screening of the monoclonal antibodies obtained by panning;

[0013] ④ Comparative analysis of antibody sequences to obtain the amino acid sequence of the monoclonal antibody G2 against cytokeratin component 19D;

[0014] ⑤ Verification of the antigen specificity of the obtained monoclonal antibody G2 against cytokeratin component 19D.

[0015] The present invention also includes the application of the above-mentioned monoclonal antibody G2 against cytokeratin component 19D in detecting cytokeratin component 19D protein.

[0016] Preferably, the application of the above-mentioned monoclonal antibody G2 against cytokeratin component 19D in detecting cytokeratin component 19D protein comprises the following steps:

[0017] ① Construction of an expression vector for the Fab fragment of the monoclonal antibody G2 against cytokeratin component 19D;

[0018] ② Expression, purification and activity verification of the Fab fragment of the monoclonal antibody G2 against cytokeratin component 19D;

[0019] ③ Preparation and activity verification of the immunofluorescence sensor G2T fluorescence quencher for detecting cytokeratin component 19D;

[0020] ④ Use of the immunofluorescence sensor G2T fluorescence quencher to detect cytokeratin component 19D.

[0021] The present invention also includes a kit for determining the concentration of cytokeratin component 19D, which contains the above-mentioned monoclonal antibody G2 against cytokeratin component 19D.

[0022] The present invention has the following advantages compared with the prior art:

[0023] By using phage display technology, panning was carried out in the human phage display antibody library Tomlinson I+J containing synthetic sequences, and a monoclonal antibody was successfully isolated. The CDR1, CDR2, and CDR3 regions of the heavy and light chains of this monoclonal antibody have novel amino acid sequences and can specifically bind to cytokeratin component 19D. This antibody can be used for the concentration detection of cytokeratin component 19D, a lung cancer marker, with high detection accuracy and good specificity. A polypeptide sequence containing cysteine (Cys-tag) was attached to the amino terminus of the antibody, and a Fab expression vector was constructed to express and purify the Fab fragment of the antibody. The antibody was labeled with a fluorescent dye containing maleimide through a maleimide-thiol reaction to prepare an immunofluorescent sensor G2T fluorescence quencher for detecting the concentration of cytokeratin component 19D, a lung cancer marker. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a result diagram of the enzyme-linked immunosorbent assay for the polyclonal antibody obtained by panning;

[0025] Figure 2 It is a result diagram of the screening of monoclonal antibody G2 against cytokeratin component 19D;

[0026] Figure 3 It is a result diagram of the competitive enzyme-linked immunosorbent assay of the monoclonal antibody;

[0027] Figure 4 It is a dose-response curve diagram of the detection of cytokeratin component 19D by the competitive enzyme-linked immunosorbent assay of the monoclonal antibody of the present invention;

[0028] Figure 5 It is the PCR amplification result of the light chain variable region and heavy chain variable region of monoclonal antibody G2 against cytokeratin component 19D;

[0029] Figure 6 It is a result diagram of the double digestion of vector pUQ2GS;

[0030] Figure 7 It is a result diagram of the colony PCR after the transformation of recombinant expression vector pUQ2GS-VL;

[0031] Figure 8 It is a result diagram of the double digestion of vector pUQ2GS-VL;

[0032] Figure 9PCR result diagram of colonies after transformation with recombinant expression vector pUQ2GS-G2;

[0033] Figure 10 Coomassie brilliant blue staining diagram of polyacrylamide gel electrophoresis for purified monoclonal antibody fragment Fab;

[0034] Figure 11 Verification result diagram of antigen-binding activity of monoclonal antibody fragment Fab;

[0035] Figure 12 Coomassie brilliant blue staining diagram of polyacrylamide gel electrophoresis for the prepared fluorescence quencher of immunofluorescence sensor;

[0036] Figure 13 Fluorescence diagram of polyacrylamide gel electrophoresis for the fluorescence quencher;

[0037] Figure 14 Verification result diagram of antigen-binding activity of the fluorescence quencher;

[0038] Figure 15 Fluorescence spectrum comparison diagram of the fluorescence quencher in buffer PBST and denaturant GdnHCl / DTT;

[0039] Figure 16 Dose-dependent fluorescence spectrum of antigen detection by fluorescence quencher G2T prepared based on monoclonal antibody G2 against cytokeratin component 19D of the present invention;

[0040] Figure 17 Dose-response curve of antigen detection by fluorescence quencher G2T prepared based on monoclonal antibody G2; Detailed implementation mode

[0041] The object of the present invention is to provide a monoclonal antibody G2 against cytokeratin component 19D, its preparation method and application. The following further describes the present invention in conjunction with specific embodiments.

[0042] Embodiment

[0043] 1. Amplification of phage display antibody library

[0044] Take 10 μL of Escherichia coli stock solution containing Tomlinson I+J phage library and add it to 20 mL of 2YT liquid medium (2YTAG) containing 1% glucose and 100 μg / mL ampicillin. Incubate the culture solution in a constant temperature shaker at 37°C and 220 rpm for 16 h until the OD600 of the bacterial solution is approximately 1.5. Subsequently, take 5 mL of this culture solution and inoculate it into 100 mL of 2YTAG medium, and continue to culture under the same conditions until OD600 reaches 0.4. Then, add 5 μL of helper phage KM13 (titer 1013 cfu / mL), and infect at 37 °C for 30 min. After the infection, place the bacterial solution at 4 °C and centrifuge at 5500 rpm for 30 min. Discard the supernatant, and centrifuge again for 10 min to completely remove the residual supernatant. Subsequently, resuspend the cells in 200 mL of 2YT liquid medium (2YTAGK) containing 0.1% glucose, 100 μg / mL Amp, and 50 μg / mL kanamycin (Kana), and culture in a constant temperature shaker at 30 °C and 220 rpm for 20 h. The next day, centrifuge the bacterial solution at 5500 rpm for 30 min, collect the supernatant, and evenly distribute it into 5 sterile 50 mL centrifuge tubes. Add 8 mL of PEG / NaCl solution (20% polyethylene glycol 6000, 2.5 M NaCl) to each tube, mix well, and place on ice bath for 1 h. Then centrifuge at 5500 rpm for 30 min, discard the supernatant, and centrifuge again for 10 min to completely remove the residual supernatant. Add 1 mL of sterile PBS buffer to each tube to dissolve the precipitate, and the resulting solution is the phage display antibody library solution. Titrate the phage display antibody library using Escherichia coli, and the results show that the concentration of the prepared antibody library is 10 13 cfu / mL.

[0045] 2. Panning of the phage display antibody library

[0046] In a 96-well microplate, add 100 μL of cytokeratin component 19D protein (antigen) diluted to 50 μg / mL with PBS to each well, and incubate overnight at 4 °C. The next day, pour out the antigen solution in the microplate wells, and add 300 μL of PBS solution containing 5% skim milk powder (MPBS) to each well to block at room temperature for 2 h. Subsequently, wash the microplate 3 times with PBST solution (PBS containing 0.1% Tween 20). Then, add to each well the phage display antibody library diluted to 10 with MPBS 1A phage display antibody library of cfu / mL was prepared. The microtiter plate was placed on a horizontal shaker and incubated at room temperature for 1 h, followed by standing at room temperature for 1 h. Then, it was washed 10 times with PBST to remove non-specifically bound phages. Trypsin powder was weighed, dissolved in PBS and formulated into a 1 mg / mL solution. 100 μL of this solution was added to each well. The microtiter plate was placed on a horizontal shaker and incubated at room temperature for 10 min, followed by standing at room temperature for 5 min to elute the phages bound to cytokeratin component 19D antigen. The eluate was collected into a 2 mL centrifuge tube and stored at 4 °C for short-term. 10 μL of Escherichia coli TG-1 was inoculated into 4 mL of 2YT liquid medium without glucose and antibiotics and cultured at 37 °C and 220 rpm until the OD600 of the bacterial solution was approximately 0.4. 100 μL of the eluted phage solution was added to 4 mL of this bacterial solution and infected at 37 °C for 30 min. Then, the bacterial solution was transferred to a centrifuge at 4 °C and centrifuged at 5500 rpm for 20 min. The supernatant was discarded. The cells were resuspended in 2YT medium containing 1% glucose and 100 μg / mL Amp (2YTAG) and cultured overnight at 37 °C and 220 rpm. The next day, 40 μL of the overnight cultured bacterial solution was taken, added to 4 mL of 2YTAG, and cultured at 37 °C and 220 rpm until OD600 was approximately 0.2. 5 μL of helper phage KM13 (titer 10 12(cfu / mL), infect at 37°C for 30 min, then place the infected bacterial solution at 4°C and centrifuge at 5500 rpm for 20 min. Discard the supernatant. Centrifuge again to completely remove the residual supernatant. Resuspend the cells in 30 mL of 2YT medium (2YTAGK) containing 0.1% glucose, 100 μg / mL Amp, and 50 μg / mL Kana, and continue culturing at 30°C in a constant temperature shaker at 220 rpm for 20 h. Centrifuge the overnight cultured bacterial solution at 4°C and 5500 rpm for 30 min, and collect the supernatant. Add 6 mL of PEG / NaCl solution to the supernatant, gently invert and mix well, and place on ice for 1 h. Subsequently, centrifuge at 4°C and 5500 rpm for 1 h, discard the supernatant, and repeat centrifugation for 10 min to completely remove the supernatant. Resuspend the precipitate in 300 μL of sterile PBS solution, and the resulting solution is the antibody library (R1) for the second round of panning. Repeat the above steps for two rounds of panning to obtain antibody libraries R2 and R3 respectively. Perform ELISA verification on the original antibody library R0 and the panned antibody libraries R1, R2, and R3 to evaluate their binding specificity to cytokeratin component 19D protein. Dilute BSA and cytokeratin component 19D to 5 μg / mL with PBS and coat them onto a 96-well microplate, 100 μL per well, 3 wells per group, a total of 4 groups, and coat overnight at 4°C. The next day, pour out the protein solution in the microplate, add 300 μL of 5% MPBS solution to each well, and block at room temperature for 2 h. Wash 3 times with PBST solution, then add 100 μL of phage library (R0, R1, R2, R3) diluted to 101 cfu with 5% MPBS solution to the 4 groups of micro wells respectively, and incubate at room temperature for 1 h. Wash 6 times with PBST, then add M13 Bacteriophage antibody (HRP), Mouse Mab diluted 1:5000, and incubate at room temperature for 1 h. Wash 12 times with PBST again, add 100 μL of TMB chromogenic solution to each well, develop color at 37°C for 5 min, and then add 50 μL of 20% hydrochloric acid solution to terminate the reaction. Use iMark TM ELISA reader (Bio-Rad) to measure the absorbance at 450 nm and 630 nm, and draw a bar graph. The results of enzyme-linked immunosorbent assay (ELISA) are as Figure 1 shown. By comparing the binding abilities of the four groups of phage display antibody libraries to cytokeratin component 19D protein, it was found that with the increase in the number of panning times, the binding ability of phage display antibody library R3 to 19D protein was significantly enhanced. In addition, the binding abilities of the four groups of phage libraries to BSA were extremely weak and showed no obvious change, indicating that the antibodies against cytokeratin component 19D in the antibody library were successfully enriched.

[0047] 3. Screening of monoclonal antibodies

[0048] Randomly select 96 single colonies from the cultured phage display antibody library R3 and inoculate them into 96-well microplates, adding 1.2 mL of 2YTAG liquid medium to each well. Subsequently, seal the microplates with sealing membranes to prevent cross-contamination caused by the overflow of the bacterial solution due to shaking. Place the microplates in a constant temperature shaker at 37 °C and incubate them overnight with shaking at a speed of 180 rpm. The next day, take 10 μL of the overnight cultured bacterial solution from each well and transfer it to a new 96-well microplate containing 90 μL of 2YTAG liquid medium. Seal it again with a sealing membrane and incubate it at 37 °C with shaking at 180 rpm until the OD600 of the bacterial solution is approximately 0.2. During this process, first mix 5 μL of helper phage KM13 (titer is 10 12 cfu / mL) thoroughly with 5 mL of 2YTAG liquid medium. Then, add it to the 96-well microplate, adding 150 μL to each well, and seal it with a sealing membrane. Place the microplate in an incubator at 37 °C for 30 min for phage infection. After the infection is completed, add 1.35 mL of 2YTAK medium without glucose to each well and seal it again. Transfer the microplate to a constant temperature shaker at 30 °C and continue to incubate it with shaking at a speed of 180 rpm for 20 h. In another 96-well microplate, add 100 μL of BSA solution diluted to 5 μg / mL with PBS and cytokeratin component 19D protein solution respectively, add the samples in a one-to-one correspondence, and coat them overnight at 4 °C.

[0049] The next day, pour out the unbound protein solution, add 300 μL of 5% MPBS solution to each well, and let it stand at room temperature for 2 h for blocking. At the same time, transfer the bacterial solution cultured for 20 h to a 2 mL centrifuge tube and centrifuge it at 4 °C and 5500 rpm for 30 min to obtain the phage supernatant. During this period, wash the 96-well microplate 3 times with PBST solution, and then add 75 μL of MPBS solution to each well. Subsequently, add 25 μL of the centrifuged phage supernatant to each well in a one-to-one correspondence, gently pipette and mix well, and incubate at room temperature for 1 h.

[0050] After incubation, wash the microplate 6 times with PBST solution, and then add M13 Bacteriophage antibody (HRP, Mouse Mab) diluted 1:5000 to each well and incubate at room temperature for 1 h. Subsequently, wash the microplate 12 times with PBST solution, add 100 μL of TMB chromogenic solution to each well, and place it in a constant temperature incubator at 37 °C for 5 min for color development. After the color development is completed, add 50 μL of 20% hydrochloric acid solution to each well to terminate the reaction. Finally, use an iMark TM microplate reader (Bio-Rad) to measure the absorbance of each well at 450 nm and 630 nm, and draw a bar chart ( Figure 2) According to the experimental results, antibodies with relatively high binding activity to cytokeratin component 19D were screened out and further verified to finally determine the positive clones.

[0051] 4. Comparative analysis of antibody sequences

[0052] Based on the above experimental results, 10 positive clones were selected, plasmids were extracted and gene sequencing was performed. An antibody named G2 was obtained. By comparing with the antibody sequences registered in the antibody gene library, no sequence identical to the antibody gene of the present invention was found. Therefore, this antibody is a novel antibody against cytokeratin component 19D protein. The detailed amino acid sequence of G2 antibody is as follows.

[0053] The G2 antibody consists of a heavy chain variable region and a light chain variable region. The heavy chain variable region includes a CDRH1 sequence, a CDRH2 sequence, and a CDRH3 sequence; the light chain variable region includes a CDRL1 sequence, a CDRL2 sequence, and a CDRL3 sequence.

[0054] The sequence of the heavy chain variable region of G2 antibody (G2-VH) is SEQ ID NO: 1; the CDRH1 sequence is SEQ ID NO: 2; the CDRH2 sequence is SEQ ID NO: 3; the CDRH3 sequence is SEQ ID NO: 4;

[0055] The sequence of the light chain variable region of G2 antibody (G2-VL) is SEQ ID NO: 5; the CDRL1 sequence is SEQ ID NO: 6; the CDRL2 sequence is SEQ ID NO: 7; the CDRL3 sequence is SEQ ID NO: 8. The specific amino acid sequence of G2 antibody is shown in Table 1.

[0056] Table 1 Amino acid sequence of G2 antibody

[0057]

[0058] 5. Antigen specificity of monoclonal antibody

[0059] In a 96-well plate, 100 μL of BSA and cytokeratin component 19D protein solution diluted to 2 μg / mL with PBS were added respectively, and the sample loading volume was 100 μL / well. Among them, 3 wells were coated with BSA and 6 wells were coated with cytokeratin component 19D protein, and the microplate was incubated overnight at 4°C. The next day, the unbound protein solution in the microplate was poured out, and 300 μL of 5% MPBS solution was added to each well and left to stand at room temperature for 2 h for blocking. Subsequently, the microplate was washed 3 times with PBST solution, and then 75 μL of MPBS and 25 μL of phage display antibody supernatant mixture were added to each well. For 3 of the wells coated with cytokeratin component 19D protein, when adding the above antibody mixture, cytokeratin component 19D protein was additionally added to make its final concentration reach 10 μg / mL for competitive reaction. Subsequently, the microplate was left to stand and incubate at room temperature for 1 h. After incubation, the microplate was washed 6 times with PBST solution, and then 1:5000 diluted anti-M13 Bacteriophage antibody (HRP, Mouse Mab) was added to each well and incubated at room temperature for 1 h. After washing the microplate 12 times, 100 μL of TMB chromogenic solution was added to each well, and the microplate was placed in a 37°C constant temperature incubator for color development for 5 min. Subsequently, 50 μL of 20% hydrochloric acid solution was added to each well to terminate the reaction. Finally, use iMark TM microplate reader (Bio-Rad) to measure the absorbance of each well at 450 nm and 630 nm, and draw a bar chart.

[0060] The experimental results are as Figure 3 shown. The G2 antibody can specifically bind to cytokeratin component 19D protein, but has no obvious binding to BSA. In addition, under the competitive condition of the presence of free cytokeratin component 19D protein, the binding ability of the G2 antibody to cytokeratin component 19D protein decreased significantly. This result indicates that the G2 antibody is a specific antibody against cytokeratin component 19D protein, and its binding is highly specific.

[0061] Dilute the PBS solution to a BSA and cytokeratin component 19D protein solution at 2 μg / mL, add 100 μL per well to a 96-well microplate, and incubate overnight at 4°C. The next day, pour off the unbound protein solution, add 300 μL of 5% MPBS solution to each well, and let it stand at room temperature for 2 h for blocking. Subsequently, wash the microplate 3 times with PBST solution. In the microplate wells coated with BSA, add the phage-displayed antibody solution diluted to 101 cfu / mL with MPBS. In the microplate wells coated with cytokeratin component 19D protein, add cytokeratin component 19D protein solutions at final concentrations of 0, 0.1, 1, 10, 100, and 1000 μg / mL respectively, and at the same time add the phage-displayed antibody solution. Set 3 replicate wells for each concentration and incubate at room temperature for 1 h. After incubation, wash the microplate 6 times with PBST solution, then add the M13 Bacteriophage antibody (HRP, Mouse Mab) diluted 1:5000 to each well and incubate at room temperature for 1 h. After washing the microplate 12 times, add 100 μL of TMB chromogenic solution to each well, and place the microplate in a 37°C incubator for color development for 5 min. Subsequently, add 50 μL of 20% hydrochloric acid solution to each well to terminate the reaction. Finally, use iMark TM ELISA reader (Bio-Rad) to measure the absorbance of each well at 450 nm and 630 nm, and draw a dose-response curve to detect the concentration of free cytokeratin component 19D in the solution by competitive ELISA method( Figure 4 ).

[0062] The experimental results show that when the concentration of free cytokeratin component 19D in the solution is low, the phage-displayed antibody mainly binds to the cytokeratin component 19D protein coated on the microplate, thus generating a higher detection signal. As the concentration of free cytokeratin component 19D protein gradually increases, the free 19D protein competes with the phage-displayed antibody for binding, resulting in a decrease in the number of antibodies bound to the microplate and thus a decrease in the detection signal. Through dose-response curve analysis, the half-inhibitory concentration (IC 50 ) of antibody G2 is determined to be 5.4 μg / mL, and the calculated lowest detection limit (LOD) of cytokeratin component 19D protein is 1.45 ng / mL.

[0063] 7. Construction of the Fab fragment expression vector of monoclonal antibody G2

[0064] Using the specific primers AgeIG2VHback / XhoIG2VHfor and SpeIG2VLback / HindIIIG2VLfor, the VH and VL gene fragments were amplified from the screened monoclonal antibody G2 respectively. The PCR reaction system and conditions were as follows: pre-denaturation at 95°C for 3 min; 34 cycles: denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s; final extension at 72°C for 5 min. The PCR products were analyzed by 1% agarose gel electrophoresis, and the results were as Figure 5 shown: single clear bands were shown at 342 bp and 324 bp in the VH and VL lanes respectively, which were consistent with the theoretical sizes, indicating successful amplification of the gene fragments. Subsequently, the target fragments were purified using a column DNA gel recovery kit. The reaction system is shown in Table 2. The purified VH and VL gene fragments were double digested with SpeI / HindIII with the pQU2GS vector respectively to generate complementary sticky ends. The digestion system is shown in Table 3. After mixing each component, centrifuge briefly for 10 s and incubate at 37°C for 16 h. The digestion products were verified by agarose gel electrophoresis ( Figure 6 ). The results showed that the digested vector formed a single linear band at about 6000 bp, which was clearly distinguishable from the undigested supercoiled plasmid (>10,000 bp), indicating complete digestion. Subsequently, the digestion products were purified using a gel recovery kit for subsequent ligation.

[0065] The double-digested VL gene fragment (2 μL) was mixed with the pQU2GS vector (3 μL) in proportion, added with LigationHigh Ver.2 ligase (5 μL), and ligated at 16°C for 2 h. The ligation products were transformed into DH5α competent cells. After ice-bathing for 30 min, heat shock at 42°C for 60 s, ice-bathe again for 10 min, and then add LB recovery medium and shake culture for 1 h. Finally, the bacterial solution was spread on LB solid medium containing 100 μg / mL ampicillin (Amp) and cultured overnight at 37°C. The next day, single colonies were randomly picked for colony PCR (the PCR reaction conditions were the same as before, and the primers are shown in Table 4 in detail). The electrophoresis results showed the target band (about 324 bp), which was consistent with the control ( Figure 7 ), indicating that the VL gene had been correctly inserted into the vector. Subsequently, positive clones were picked and inoculated into 4 mL LB liquid medium (containing 100 μg / mL Amp), and cultured with shaking at 37°C and 250 rpm until OD600≈1.5. After plasmid extraction, the concentration was measured using Nanodrop and sent for sequencing verification. The recombinant plasmid with correct sequencing results was named pQU2GS-VL.

[0066] The AgeI / XhoI double-digested and purified VH gene fragment was ligated with the pQU2GS-VL plasmid. After confirming the linearization of the vector by electrophoresis ( Figure 8) According to the above connection and transformation steps, the VH fragment was inserted into the vector. The results of colony PCR (primers: T7 promoter / T7 terminator) showed a target band (about 342 bp), which was consistent with the expected size. Figure 9 ) Subsequently, sequencing verification was carried out, and finally the Fab expression vector was constructed and named pQU2GS-G2. The primer sequences and vector construction strategy are shown in Table 5.

[0067] Table 2 PCR reaction system

[0068]

[0069] Table 3 V L gene and pQU2GS vector digestion system

[0070]

[0071] Table 4 Colony PCR reaction system

[0072]

[0073]

[0074] Table 5 Primer sequences for amplifying target genes

[0075]

[0076] 8. Expression, purification and activity verification of Fab fragment of G2 antibody

[0077] The recombinant vector pUQ2GS-G2 was transformed into Escherichia coli competent cells SHuffle T7 Express lysY. Subsequently, the bacterial solution was evenly spread on an LBA plate and cultured overnight at 37 °C. The next day, single colonies were picked for colony PCR screening, and positive clones were inoculated into 4 mL of LBA liquid medium and cultured overnight at 37 °C and 250 rpm. 3 mL of the overnight culture was inoculated into 300 mL of LBA liquid medium and cultured with shaking at 37 °C and 250 rpm until the OD600 reached about 0.5. At this time, 150 μL of 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and induction expression was carried out at 16 °C and 150 rpm for 20 h.

[0078] After the culture was completed, centrifugation was carried out at 4 °C and 12,000 rpm for 5 min, and the cell pellet was collected and resuspended followed by ultrasonic disruption. Subsequently, centrifugation was carried out at 4 °C and 12,000 rpm for 10 min, and the supernatant was collected. Take 500 μL of Ni-NTA Sefinose TMPlace the resin in a 2 mL centrifuge tube, add 1 mL of washing buffer, mix by shaking, centrifuge at 800 rpm for 1 min, and discard the supernatant. Mix the washed resin with the protein supernatant and incubate on a shaker at 4°C with slow rotation for 2 h to allow the target protein to bind fully to the affinity resin. Transfer the bound mixture to a sonicated purification column and collect the flow-through to prevent incomplete binding of the target protein. Wash with 20 mL of washing buffer containing 10 mM imidazole to remove non-specifically bound proteins. Then, elute the target protein with 3 mL of elution buffer containing 250 mM imidazole, adding 1 mL each time and allowing it to flow out naturally, repeating 2 times, and collecting 3 eluates in total. Immerse the eluted affinity resin in 10 mL of elution buffer for 20 min and wash with 20 mL of washing buffer containing 5 mM imidazole, and finally store it in a 20% ethanol solution at 4°C for future use.

[0079] The collected protein samples were measured for concentration using NanoDrop, and 5 μL was taken for SDS-PAGE electrophoresis analysis to evaluate the molecular weight and purity of the protein. As Figure 10 shown, two distinct bands appeared between 25 - 35 kDa, corresponding to the heavy chain (VH-CH1, theoretical molecular weight 27.7 kDa) and light chain (VL-CL, theoretical molecular weight 26.9 kDa) of the antibody Fab fragment. The experimental results were basically consistent with the theoretical values, indicating that the recombinant G2 antibody Fab fragment with high purity was successfully obtained.

[0080] The antigen-binding activity of the Fab fragment of the G2 antibody was evaluated by enzyme-linked immunosorbent assay (ELISA). A 96-well microplate was coated with 5 μg / mL cytokeratin component 19D and bovine serum albumin (BSA) solutions, 100 μL per well, with 3 replicates per group, and incubated overnight at 4 °C. The next day, it was blocked with 5% non-fat milk PBS (MPBS) solution and left standing at room temperature for 2 h. Subsequently, it was washed 3 times with PBST, and 100 μL of 10 μg / mL G2 antibody Fab solution was added to each well and incubated at room temperature for 1 h. After washing 6 times, 1:3000 diluted Myc-Tag Mouse mAb was added to each well as the secondary antibody and incubated at room temperature for 1 h. After washing 6 times again, 1:3000 diluted HRP-conjugated Rabbit anti-mouse IgG was added to each well and incubated at room temperature for 1 h. After washing 12 times, 100 μL of TMB chromogenic solution was added to each well and incubated at 37 °C for 5 min. Subsequently, 50 μL of 15% hydrochloric acid was added to each well to terminate the reaction, and the absorbance was measured at 450 nm using a microplate reader. The absorbance at 630 nm was used as the background correction data, and a bar chart was plotted.

[0081] As Figure 11 shown, the absorbance of the experimental group samples coated with cytokeratin component 19D was 0.8, while the absorbance of the control group samples coated with BSA was 0.07, and there was a significant difference in absorbance between the two groups. This result indicates that the expressed and purified Fab fragment of the G2 antibody has high antigen-binding specificity and can be used for further functional studies.

[0082] 9. Preparation of the fluorescence quencher G2T of the immunofluorescence sensor

[0083] In this experiment, the Fab fragment of the G2 antibody was labeled with the fluorescent dye 5(6)-carboxytetramethylrhodamine (TAMRA) to prepare the fluorescence quencher G2T, and the entire labeling process was carried out under light-proof conditions.

[0084] Dilute the purified antibody Fab to 1 mg / mL. Take 100 μL and place it in a 2 mL centrifuge tube. Add 0.2 μL of 1 M tris(2-carboxyethyl)phosphine hydrochloride (TCEP) to make the final concentration 0.5 mM. Incubate at 4 °C on a vertical shaker in the dark with rotation for 20 min to mildly reduce the exposed thiol groups of the Fab fragment. Subsequently, add a 4-azidobenzoic acid (ABA) solution with a final concentration of 2 mM. Invert and mix well, then let it stand on ice for 10 min to terminate the reaction. Next, add 1 μL of the fluorescent dye TAMRA and incubate at 4 °C in the dark on a shaker for 4 h for fluorescence labeling. After the labeling is completed, mix the reaction solution with 100 μL of Anti-DYKDDDDK G1 Affinity Resin (anti-FLAG tag affinity resin) and 1 mL of TBS buffer, and incubate on a shaker at 4 °C in the dark for 1 h to achieve binding. After incubation, transfer the solution to a purification column and gently squeeze out the solution using an ear pipette bulb. Subsequently, wash with 20 mL of TBS to remove the free fluorescent dye. Finally, add 200 μL of a 3×Flag polypeptide solution with a concentration of 150 μg / mL, incubate at room temperature for 10 min, and collect the eluate, which is the prepared fluorescence quencher, named G2T fluorescence quencher.

[0085] The prepared fluorescence quencher was analyzed by SDS-PAGE. Observe the fluorescence signal under a gel imaging analysis instrument (as Figure 12 shown), and then stain with Coomassie Brilliant Blue (CBB) staining solution. The staining result is as Figure 13 shown. The CBB staining pattern shows that there are two bands between 25 - 35 kDa, corresponding to the heavy chain and light chain of the antibody Fab fragment respectively. Comparing Figure 12 and Figure 13 it can be seen that the TAMRA fluorescent dye was successfully labeled on the heavy chain of the Fab fragment, indicating that the G2T fluorescence quencher was successfully prepared.

[0086] The antigen-binding activity of the G2T fluorescence quencher was evaluated by enzyme-linked immunosorbent assay (ELISA). In the experiment, the G2T fluorescence quencher was used to replace the traditional monoclonal antibody Fab as the primary antibody, and the experiment was carried out according to the detection procedure for the antigen-binding activity of monoclonal antibody Fab to ensure that the labeling of the fluorescent dye does not affect the antigen-binding ability of the antibody.

[0087] The experimental results (as Figure 14As shown, the absorbance of cytokeratin component 19D was 0.84, while that of bovine serum albumin (BSA) was only 0.07. This result proved that the TAMRA fluorescence labeling did not significantly affect the antigen-binding activity of the G2T fluorescence quencher, indicating that the fluorescence quencher still maintained good biological functions and could be used for subsequent immunofluorescence detection.

[0088] 10. Immunofluorescence sensor G2T fluorescence quencher for detecting cytokeratin component 19D

[0089] To evaluate the fluorescence dequenching effect of the G2T fluorescence quencher, that is, the ability of the fluorescent dye to be effectively quenched by tryptophan in the antibody to form a fluorescence quencher, a fluorescence release experiment was carried out using the denaturant GdnHCl / DTT. The specific steps were as follows: Equal amounts of the G2T fluorescence quencher were added to 700 μL of PBS buffer and 700 μL of the denaturant GdnHCl / DTT (7 M guanidine hydrochloride and 100 mM dithiothreitol, with PBS as the solvent), and incubated in the dark for 30 min. Subsequently, the incubated solution was added to a black opaque 96-well microplate, 200 μL per well, with 3 replicates in each group, and the fluorescence intensity at 550 - 700 nm (TAMRA) was scanned using a SpectraMax i3x multi-functional microplate reader, with the excitation wavelength set at 480 nm. The experimental results showed that compared with the PBS buffer, the peak fluorescence intensity increased by 3.04 times after treatment with GdnHCl / DTT (as Figure 15 shown), indicating that the fluorescent dye in the G2T fluorescence quencher was successfully quenched and could be released by the denaturant, thus verifying the feasibility of this fluorescence quenching system.

[0090] To evaluate the sensitivity of the G2T fluorescence quencher in detecting cytokeratin component 19D, the experiment was designed as follows: 1) Take 9 centrifuge tubes, with 1 as a blank control, adding 700 μL of PBS buffer and 1 μL of the G2T fluorescence quencher. 2) The remaining 8 centrifuge tubes were respectively added with 700 μL of cytokeratin component 19D at different concentrations (1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL) and 1 μL of the G2T fluorescence quencher. 3) After incubating in the dark for 30 min, the incubation solution was transferred to a black opaque microplate, 200 μL per well, with 3 replicates in each group, and the fluorescence intensity was measured.

[0091] The experimental results are as Figure 16As shown, the fluorescence signal reaches its maximum peak at 580 nm, which is consistent with the fluorescence emission spectrum of the TAMRA dye. At the same time, the fluorescence intensity gradually increases with the increase in the concentration of cytokeratin component 19D. When the concentration increases to 10 μg / mL, the fluorescence intensity reaches 1.99 times the initial fluorescence intensity, indicating that the G2T fluorescence quencher can be used for in vitro detection of cytokeratin component 19D.

[0092] To further evaluate the sensitivity of this detection method, the fluorescence intensity at 575 nm was normalized (n = 3), and a dose-response curve was plotted by inserting a standard curve (as Figure 17 shown). The calculated limit of detection (LOD) of the G2T fluorescence quencher for cytokeratin component 19D was 2.41 pg / mL, the half-maximal effective concentration (EC50) was 1.37 ng / mL, and the linear detection range was 10 pg / mL - 100 ng / mL. This indicates that this method has high detection sensitivity and a wide linear range in the detection of cytokeratin component 19D, providing strong technical support for the detection of related biomarkers.

Claims

1. A monoclonal antibody G2 against cytokeratin component 19D, characterized in that: The monoclonal antibody G2 against cytokeratin component 19D specifically binds to the cytokeratin component 19D protein; the monoclonal antibody G2 against cytokeratin component 19D comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementary determining regions, namely CDRH1, CDRH2 and CDRH3; the light chain variable region comprises three complementary determining regions, namely CDRL1, CDRL2 and CDRL3; The amino acid sequence of the heavy chain variable region of the monoclonal antibody G2 against cytokeratin component 19D is SEQ ID NO: 1, the amino acid sequence of CDRH1 is SEQ ID NO: 2, the amino acid sequence of CDRH2 is SEQ ID NO: 3, and the amino acid sequence of CDRH3 is SEQ ID NO: 4; The amino acid sequence of the light chain variable region of the monoclonal antibody G2 against cytokeratin component 19D is SEQ ID NO: 5, the amino acid sequence of CDRL1 is SEQ ID NO: 6, the amino acid sequence of CDRL2 is SEQ ID NO: 7, and the amino acid sequence of CDRL3 is SEQ ID NO:

8.

2. A method for preparing a monoclonal antibody G2 against cytokeratin component 19D, characterized in that Comprising the following steps: ① Amplification of the Tomlinson I+J phage display antibody library; ② Panning of the Tomlinson I+J phage display antibody library; ③ Screening of the monoclonal antibodies obtained by panning; ④ Comparative analysis of antibody sequences to obtain the amino acid sequence of the monoclonal antibody G2 against cytokeratin component 19D; ⑤ Verification of the antigen specificity of the obtained monoclonal antibody G2 against cytokeratin component 19D.

3. Use of the monoclonal antibody G2 against cytokeratin component 19D according to claim 1 in detecting cytokeratin component 19D protein.

4. Use of the monoclonal antibody G2 against cytokeratin component 19D according to claim 3 in detecting cytokeratin component 19D protein, characterized in that: Comprising the following steps: ① Construction of an expression vector for the Fab fragment of the monoclonal antibody G2 against cytokeratin component 19D; ② Expression, purification and activity verification of the Fab fragment of the monoclonal antibody G2 against cytokeratin component 19D; ③ Preparation of the immunofluorescent sensor G2T fluorescence quencher for detecting cytokeratin component 19D and its activity verification; ④ Use of the immunofluorescent sensor G2T fluorescence quencher for detecting cytokeratin component 19D.

5. A kit for determining the concentration of cytokeratin component 19D, characterized in that: Comprising the monoclonal antibody G2 against cytokeratin component 19D according to claim 1.

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