Anti-topo ii alpha protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human Topo II alpha antigen, secretes cell strain, its preparation method and its use in immunodetection.The above technical solution selects the amino acid of 1301-1531 of Topo II alpha protein as antigen peptide, codon optimization becomes the gene fragment suitable for expression in escherichia coli BL21, and finally the recombinant protein includes Topo II alpha protein fragment and histidine protein label.The recombinant protein is immunized to mouse, is fused by cell, screening and subcloning, and obtains mouse hybridoma cell strain, and the anti Topo II alpha protein monoclonal antibody secreted by the cell strain is obtained.The antibody obtained by the present application has high specificity, sensitivity, and can specifically recognize the cell expressing Topo II alpha protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to an anti-Topo Ⅱα protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] Topo Ⅱα, i.e. topoisomerase Ⅱα, is an enzyme that plays a key role in the process of DNA replication, transcription and repair. DNA topoisomerase is a kind of essential nuclear enzyme widely existing in organisms, which can change the spatial structure state of DNA without causing changes in its chemical structure. Topoisomerase (Topo) is divided into I and II types, and there are two isozymes of Topo Ⅱ in humans, namely Topo Ⅱα and Topo Ⅱβ. Among them, Topo Ⅱα is closely related to cell drug resistance, and Topo Ⅱα is located on chromosome 17 with a molecular weight of 170KD.

[0003] The expression level of Topo Ⅱα in various tumor tissues is significantly higher than that in normal tissues, which makes it one of the important markers for tumor diagnosis and prognosis evaluation. The positive expression rate of Topo Ⅱα in breast cancer tissue is significantly higher than that in normal breast tissue. In particular, in breast invasive ductal carcinoma, the positive expression rate of Topo Ⅱα is higher, and is related to lymph node metastasis and high expression of HER-2 and Ki-67. In gastric cancer tissue, Topo Ⅱα also presents a high expression state, and the expression level is related to the differentiation degree of gastric cancer tissue. The positive rate of Topo Ⅱα in low differentiated gastric cancer is significantly higher than that in high and medium differentiated gastric cancer. In addition to breast cancer and gastric cancer, Topo Ⅱα also has different degrees of expression in other types of tumors, such as lung cancer, colorectal cancer, etc. The expression level of Topo Ⅱα is closely related to the malignant degree, metastatic potential and prognosis of tumors. Therefore, detecting the expression level of Topo Ⅱα can provide an important basis for the prognosis evaluation of tumors. For example, in breast cancer, high expression of Topo Ⅱα often indicates poor prognosis. SUMMARY

[0004] The present application provides an anti-Topo Ⅱα protein monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0005] Further, the monoclonal antibody is a murine monoclonal antibody, and the clone number is 25F11.

[0006] The present application further provides a preparation method of an anti-Topo Ⅱα protein monoclonal antibody, and the antigen for immunizing mice is a recombinant protein, which is expressed by E. coli.

[0007] Further, the recombinant protein comprises Topo II alpha protein fragment and HIS protein tag.

[0008] Further, the Topo II alpha protein fragment is the fragment of 1301-1531 sites, which is the amino acid sequence shown in SEQ ID NO. 3.

[0009] Further, the plasmid vector selected in the recombinant process is pET30a.

[0010] The inventor also provides a Topo II alpha protein immunodetection reagent, which contains the above-mentioned anti-Topo II alpha protein monoclonal antibody as an effective component.

[0011] Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunization.

[0012] Differing from the prior art, the beneficial technical effects of the present application are that the above-mentioned technical solution selects the amino acid of 1301-1531 sites of Topo II alpha protein as antigen peptide, carries out codon optimization, becomes a gene fragment suitable for expression in Escherichia coli BL21 (DE3), and finally obtains a recombinant protein comprising Topo II alpha protein fragment and histidine protein tag. The recombinant protein is used for immunizing mice, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain 25F11 secreting anti-Topo II alpha protein monoclonal antibody is obtained, and the anti-Topo II alpha protein monoclonal antibody secreted by the cell strain. The antibody obtained by the present solution has high specificity and sensitivity, can specifically recognize cells expressing Topo II alpha protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a comparison chart of immunohistochemical staining results of breast cancer (left: Topo II alpha prepared by the present application, right: commercially available Topo II alpha).

[0014] Figure 2 It is a comparison chart of immunohistochemical staining results of testicular tissue (left: Topo II alpha prepared by the present application, right: commercially available Topo II alpha). DETAILED DESCRIPTION

[0015] In order to explain the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0016] The term "embodiment" is mentioned in the present application means that the specific features, structures or properties described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0017] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms herein is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0018] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in the present application generally represents that the associated objects before and after are a "or" logical relationship.

[0019] In the present application, the terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0020] In the present application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0021] As the same understanding as in the "Guidelines for Examination", in the present application, the expressions such as "greater than", "less than", "exceed" are understood as not including the number; the expressions such as "above", "below", "within" are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0022] Preparation of recombinant Topo IIa protein fragment of Example 1

[0023] I. Gene optimization and synthesis

[0024] Topo IIa according to the protein sequence of accession number NP_001058.2 in NCBI database, the protein fragment of 1301-1531 amino acids was selected and directly optimized into a gene fragment suitable for expression in E. coli BL21 (DE3). BamH I and Xho I enzyme cutting sites were added at the 5' and 3' ends of the gene during PCR.

[0025] The PCR product was recovered after agarose gel electrophoresis separation, and the recovered fusion protein gene and the plasmid vector pET30a for expression were subjected to BamH I and Xho I enzyme cutting, respectively, and then recovered after electrophoresis again, and linked with T4 DNA ligase. The ligation product was transformed into competent cells of E. coli BL21 (DE3), and the clones on the plate were inoculated and subjected to bacterial liquid PCR identification. The clones with positive PCR results were selected for sequencing analysis, and the clones with completely correct sequences were used.

[0026] Different antigens were selected for immunization, which might prepare antibodies with different binding properties. The molecule simultaneously exists in a variety of variants caused by variable splicing, which eventually leads to different abilities and modes of different antibodies to recognize cells expressing antigens. According to the published sequence, the Topo IIa molecule was analyzed, and based on its structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, a suitable region for soluble expression and good immunogenicity was selected for recombinant expression. The amino acid residues of 1301-1531 of Topo IIa were subjected to codon optimization, and the molecular weight was about 170 kDa. The Topo IIa protein was obtained by sequence optimization design using prokaryotic expression gene sequence. The recombinant immunogen was composed of the Topo IIa protein fragment with antigenicity and the protein tag for purification of the recombinant protein, and the protein tag was HIS.

[0027] II. Protein expression and purification

[0028] The single colony culture overnight bacteria were transferred to 100 mL LB medium at a ratio of 1:100, kanamycin was added at a final concentration of 10 μg / mL, and the bacteria were cultured at 37°C with shaking until the OD600 was 0.6-0.8. Then, 0.5 mmol / L IPTG was added, and the bacteria were cultured at 16°C with shaking overnight. After collecting the bacteria, ultrasonic disruption was performed. The recombinant protein with a histidine tag was subjected to affinity purification using a nickel column. Elution was performed using 500 mmol / L imidazole, and SDS PAGE separation and detection were performed. The concentration of the recombinant Topo IIa protein with a fusion histidine tag was 0.5 mg / mL, which could be used for animal immunization and antibody screening and identification.

[0029] Example 2 Establishment of hybridoma cell lines

[0030] I. Immunization

[0031] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week old female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection of 6 points per mouse at a dose of 20 μg per mouse. The mice were boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. Seven days after the third boost, the serum of the mice was tested for the titer of anti-antigen polyclonal antibodies by indirect ELISA (wavelength 450 nm). The mouse with the highest titer was given a boost by tail vein injection of the antigen mixed with normal saline at a dose of 20 μg per mouse.

[0032] II. Cell fusion

[0033] The spleen cell suspension of the immunized mice was prepared aseptically and mixed with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded, and the centrifuge tube was placed in a 37°C water bath. Within 1 min, 1 mL of PEG1500 (Roche) was slowly added and the cells were stirred. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10 mL of serum (PAA) was carefully added to the cells, which were blown up, and 5 mL of 10x HAT (Sigma) mixed with thymocytes was added, mixed well. Then, 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed well, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in a 37°C 5% CO2 incubator.

[0034] III. Cloning and ELISA screening of positive hybridoma cells (25F11)

[0035] Seven days after fusion, the size and density of the cell colonies were moderate. Under a dissecting microscope, round, solid, and large colonies were sucked into 96-well culture plates containing medium, and cultured in a 37°C 5% CO2 incubator. After 3 days, the cells occupied about 2 / 3 of the bottom area, and 100 μL of supernatant was taken for ELISA screening with the immunogen and synthetic polypeptide, respectively. The positive clones were completely changed, and 200 μL of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening was performed, and the positive clones were transferred to 24-well plates containing medium (containing feeder cells and HT) for culture. Five days later, 100 μL of supernatant was taken for the third ELISA screening, and the positive clones were successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

[0036] I. Preparation of ascites

[0037] Log phase hybridoma cells were washed with serum-free medium and suspended, and about 5 x 105cells were injected intraperitoneally into each of 5 mice. 5 , 1 mL. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites were collected 7 days later. The removed ascites were centrifuged at 4000 rpm for 10 min at 4°C. The middle ascites were carefully sucked out and collected in a centrifuge tube, which was stored at 4°C or -20°C.

[0038] II. Purification of Monoclonal Antibody

[0039] The antibody was purified from the ascites by affinity chromatography with HiTrap rProtein A FF (GE) according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by the Bradford method. The purified antibody was stored at -20°C.

[0040] Example 4 Characterization of Monoclonal Antibody

[0041] I. Subclass Identification

[0042] Sheep anti-mouse IgG (Beijing Zhongshanjinqiao Biotechnology Co., Ltd.) was diluted to 0.5 μg / mL with 100 mM PBS (pH 7.4), 100 μL was added to each well, and incubated at 4°C overnight. The liquid was poured out, and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200 μL of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 0.1 mL of HRP-labeled sheep anti-mouse (κ, λ) antibody or 1:2000 diluted HRP-labeled sheep anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) was added to the appropriate wells, respectively, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 50 μL of citric acid buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 was added to each well for color development, and the OD value at 405 nm was determined within 10-20 min.

[0043] The results show that the monoclonal antibody of the present application is an IgG1 type mouse monoclonal antibody.

[0044] II. Affinity Constant Determination

[0045] Coat Topo IIa recombinant protein prepared in Example 3, coating concentration is 2 μg / mL, 100 μL / well, 4°C coating overnight, PBS-T wash 3 times. Add 200 μL blocking solution to each well, 37°C blocking for 2 hours, PBS-T wash 3 times. The monoclonal antibody purified in Example 3, starting from 1:200, 2-fold gradient dilution, the last one well left blank control, 37°C incubation for 1 hour, PBS-T wash 3 times. HRP labeled goat anti-mouse secondary antibody 1:20000 dilution, 100 μL per well, 37°C incubation for 1 hour, PBS-T wash 3 times. Add 100 μL of 0.1% TMB (Sigma) and 0.03% H2O2 containing citric acid-phosphoric acid buffer solution to each well, color for 10 minutes, add 50 μL of 0.5M sulfuric acid solution to stop the reaction. Measure the absorbance value at 450 nm with a microplate reader. Draw the OD value corresponding to the antibody dilution curve, find the dilution corresponding to half of the maximum binding OD value A, and calculate the affinity constant of the antibody as 1.92 x 10 9 .

[0046]

[0047] III. Monoclonal antibody reaction specificity and application effect

[0048] Take the Topo IIa recombinant protein prepared in Example 1, and use the immunoblotting method to detect the recognition specificity of the monoclonal antibody of the application, and perform 12% polyacrylamide gel electrophoresis. The gel protein band is transferred to a PVDF membrane (Millipore Corporation) in a Bio-Rad electrotransfer system according to the conventional method. The membrane is placed in a TBS-T blocking solution containing 5% skim milk at 4°C overnight. Add the Topo IIa protein monoclonal antibody purified in Example 3 and incubate at 4°C overnight. After washing the membrane with TBS-T, add goat anti-mouse secondary antibody (Beijing Zhongshen Jinqiao Biotechnology Co., Ltd.) diluted 1:5000, and incubate at room temperature for 1 hour. Wash the membrane again with TBST, add ECL ultra-sensitive color developing liquid (Beijing Puli Lei Gene Technology Co., Ltd.), and collect the chemiluminescence image data with a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

[0049] Example 5: Determination of the variable region sequence of the antibody

[0050] Take fresh hybridoma cells for culture, and take the supernatant for antigen binding property verification, to confirm that the cell strain used for cloning can indeed secrete the required antibody. After the result is confirmed, centrifuge to collect 10 6The hybridoma cells were used. Total RNA of the hybridoma cells was extracted by Trizol method. 9 μL of total RNA, 2.5 μL of oligo(dT)12-18 primer (10 mM), and 5 μL of dNTPs were mixed, and then incubated at 70 °C for 5 min and on ice for 5 min, or denatured according to the reverse transcriptase used. Then, 5 μL of RT buffer (5X), 2.5 μL of DTT (0.1 M), and 1 μL of reverse transcriptase were added, and the mixture was incubated at 42 °C for 1 h. The reaction was terminated by incubation at 70 °C for 15 min, and the obtained cDNA was stored at -20 °C. The obtained first-strand cDNA was subjected to PCR amplification. In a 50 μL reaction system, 25 pmol of each primer was added, and the sequences of the primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the primer sequences for mouse monoclonal antibodies described in the book "Recombinant Antibodies" (Science Press, published in 2005) edited by Shen Bei-fen.

[0051] The remaining dNTPs and buffers were added according to the conventional method, and finally, 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set as 94 °C for 40 s, 52 °C for 40 s, and 72 °C for 40 s, and 20 to 25 cycles were performed, and finally, 72 °C was extended for 3 min. The product was stored at 4 °C or directly subjected to electrophoresis. 20 μL of the PCR product was subjected to electrophoresis analysis, and separated on a 1.5% agarose gel. The length of the light chain (κ light chain) was between 320-340 bp, and the length of the heavy chain was between 340-370 bp. When the specific product in this region was obtained, the gel was cut and recovered, and then cloned into a T vector or an expression vector for sequencing.

[0052] Example 6. Immunohistochemical tissue chip staining and identification

[0053] I. Chip preparation process

[0054] Each sample was first subjected to HE section staining to determine the tumor site. The full-automatic tissue chip instrument of 3DHISTECH Company was used to prepare the tissue chip. The prepared tissue chip wax block was placed into a wax block preparation mold, and then placed into a 68 °C oven for 10 min to make the tissue chip and the wax of the receptor wax block into one body. Then, the mold was gently taken out of the oven, and the semi-melted paraffin was cooled at room temperature for about 30 min. Then, the tissue chip wax block was placed into a -20 °C refrigerator for freezing for 6 min, and then taken out of the mold. The tissue chip was sectioned or stored in a 4 °C refrigerator for standby use. After trimming, continuous sections were prepared with a thickness of 3 μm. The continuous sections were floated in 40% alcohol to naturally expand, and then transferred to warm water at 50 °C for 30 s. The sections were attached to glass slides treated with polylysine. The prepared tissue chip was placed into a 68 °C oven for 2 h for sectioning, and then taken out, cooled at room temperature, and stored in a -4 °C refrigerator.

[0055] II. IHC staining and analysis

[0056] Routine dewaxing of xylene 3 times, 6 minutes each, 100%, 100%, 95%, 85% gradient ethanol hydration, 3 minutes each, last tap water rinse. Antigen retrieval was performed, then the sections were placed in a wet box and rinsed in PBS 3x3 minutes. 3% H2O2 was added and incubated for 10 minutes, rinsed in PBS 3x3 minutes. The sections were spun down, the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody to dilute the antibody ratio) and incubated at room temperature (25°C) for 1 hour, rinsed in PBS 3x3 minutes, the secondary antibody was added and incubated at room temperature for 15-30 minutes, rinsed in PBS 3x3 minutes, the PBS was spun off, and fresh DAB developing solution was added to develop for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and PBS was returned to blue for 30 seconds. Sequential dehydration was performed according to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing for 3 minutes, neutral resin mounting.

[0057] The results of immunohistochemical staining are divided into: positive and negative. Positive expression must be at the site of cell and tissue-specific antigen to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

[0058] 1. The sample is weakly positive; marked as "+";

[0059] 2. The sample is moderately positive; marked as "++";

[0060] 3. The sample is highly positive; marked as "+++".

[0061] 4. The sample is negative, marked as "-".

[0062] III. Data statistics

[0063] 1. Tumor tissue chip detection results:

[0064] The antibody Topo II alpha (25F11) prepared in the present application and the commercially available antibody Topo II alpha (Ki-S1) were used to simultaneously detect 46 cases of cervical squamous cell carcinoma and 40 cases of breast cancer and compare the detection results.

[0065] The immunohistochemical results of Topo II alpha were statistically analyzed. The entire test process was designed with double-blind, and the statistical results are as follows:

[0066]

[0067] The results show that the prepared anti-Topo Ⅱ alpha protein monoclonal antibody has accurate staining localization, clear staining and no non-specific staining, and a clean background. In the immunohistochemical detection, the positive rate is equivalent to that of the commercial antibody, and the positive intensity of 2 cases is higher than that of the commercial antibody. It is indicated that the Topo Ⅱ alpha (25F11) has higher sensitivity and effectively avoids false negative results.

[0068] Figure 1 A breast cancer immunohistochemical staining result comparison chart (left: Topo Ⅱ alpha prepared by the application, right: commercial Topo Ⅱ alpha).

[0069] 2. Normal tissue chip detection result:

[0070] The normal tissue chip includes 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0071] The prepared antibody (25F11) and the commercial antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, which indicates that the specificity of the prepared antibody in the normal tissue is equivalent to that of the commercial antibody. Figure 2 A testicular tissue immunohistochemical staining result comparison chart (left: Topo Ⅱ alpha prepared by the application, right: commercial Topo Ⅱ alpha).

[0072] Finally, it should be noted that although the above-mentioned embodiments have been described in the specification and drawings of the application, the patent protection scope of the application should not be limited. Any equivalent structure or equivalent flow replacement or modification based on the essential concept of the application, the contents described in the specification and drawings, and the implementation of the technical solutions of the above embodiments in other related technical fields directly or indirectly, are all included in the patent protection scope of the application.

Claims

1. An anti-Topo IIa protein monoclonal antibody, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a murine monoclonal antibody.

3. A Topo IIα protein immunoassay reagent, characterized by, The immunodetection reagent contains the anti-Topo II alpha protein monoclonal antibody of claim 1 as an effective component.

4. The immunoassay test reagent of claim 3, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

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