A CD34 biofusion enzyme antibody with a signal protein tag and its application

Abstract

This invention discloses a CD34 biofusion enzyme antibody with a signal protein tag and its applications. The amino acid sequence of the light chain variable region of the anti-human CD34 antibody is shown in SEQ ID NO: 11, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7. A signal protein tag HRP is inserted at the N-terminus of the light chain of the anti-human CD34 antibody, resulting in an HRP-CD34 antibody with a light chain variable region as shown in SEQ ID NO: 9. Furthermore, this invention provides a method for preparing the HRP-CD34 antibody. The HRP-CD34 antibody is a molecule that is completely secreted along with the antibody, exhibiting no heterogeneity, high sensitivity and specificity, and stable performance. It eliminates the need for a secondary antibody during detection, thus saving costs and improving efficiency.

Description

Technical Field

[0001] This invention relates to the field of biological disease detection technology, specifically to a CD34 biofusion enzyme antibody with a signal protein tag and its application. Background Technology

[0002] CD34 is a single-stranded transmembrane glycoprotein with a relative molecular mass of 110,000 encoded by a gene located at 1q32. It is expressed in normal endothelial cells, splenic marginal zone cells, and dendritic stromal cells surrounding blood vessels, nerves, muscle bundles, skin appendages, and the matrix of mammary lobules. CD34 is positively expressed in vascular tumors. It is also expressed in the following tissues: immature leukemias (including AML, ALL, and granulocytic sarcoma), lymphoblastic lymphoma, transformed myelodysplastic syndrome, smooth muscle cells and their derived tumors, gastrointestinal stromal tumors, schwannomas, lipomas, and epithelioid sarcomas. Benign and malignant fibrous histiocytomas and fibrosarcomas are rarely positive for CD34. Sarcomatoid mesothelioma, synovial sarcoma, and fibromatosis do not express CD34. CD34 is mainly used to determine whether there is endothelial cell differentiation. When used in combination with CD117, it is used to identify gastrointestinal stromal tumors, differentiate between simple fibrous tumors (usually positive) and sarcomatoid mesothelioma (usually negative), dermatofibrosarcoma protuberans (usually positive) and benign fibrous histiocytoma (usually negative), spindle cell carcinoma of the breast (CD34-) and malignant phyllodes tumor (CD34+).

[0003] However, commonly used CD34 immunohistochemistry kits have the following drawbacks: 1. The required staining time is relatively long, which cannot meet the needs of rapid intraoperative pathological diagnosis in clinical practice, thus limiting its application. It can only be used for auxiliary pathological diagnosis of routine paraffin sections, and not for auxiliary pathological diagnosis of intraoperative frozen sections. 2. Signal detection is achieved by chemically conjugating HRP and other signaling enzymes to CD34 antibodies. The conjugation efficiency is difficult to control, leading to significant differences in antibody staining results between different batches; in addition, the conjugation yield is very low, resulting in significant antibody waste. Therefore, there is an urgent clinical need for an immunohistochemistry kit for CD34 target detection that offers short staining time and accurate and stable staining results. Summary of the Invention

[0004] To address the above problems, the present invention provides an HRP-CD34 antibody, which is a CD34 biological fusion enzyme antibody with the signal protein tag HRP, and the signal protein tag HRP is completely secreted along with the CD34 antibody.

[0005] An anti-human CD34 antibody, wherein the amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 11 or a sequence having more than 80% sequence similarity to SEQ ID NO. 11; and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7 or a sequence having more than 80% sequence similarity to SEQ ID NO. 7.

[0006] The nucleotide sequence expressing the anti-human CD34 antibody is described.

[0007] A CD34 biofusion enzyme antibody with a signal protein tag, wherein the antibody is formed by inserting a signal protein tag at the N-terminus of the light chain of the aforementioned anti-human CD34 antibody; the signal protein tag is horseradish peroxidase (HRP).

[0008] The amino acid sequence of the light chain variable region of the CD34 biofusion enzyme antibody with signal protein tag is shown in SEQ ID NO: 9.

[0009] The nucleotide sequence encoding the CD34 biofusion enzyme antibody with the signal protein tag.

[0010] An expression vector containing the above-described nucleotide sequence.

[0011] Recombinant host cells containing the above expression vectors.

[0012] A method for preparing the CD34 biofusion enzyme antibody with a signal protein tag, comprising:

[0013] 1) The recombinant host cells are cultured under suitable conditions to express antibodies;

[0014] 2) Then the antibodies are purified and collected from the host cells.

[0015] The application of the anti-human CD34 antibody and the CD34 biofusion enzyme antibody with signal protein tag in the preparation of CD34 target immunohistochemical detection kit. Beneficial effects

[0016] The advantages of this invention, which differ from existing technologies, are as follows:

[0017] ① HRP-CD34 antibody carries the signal protein tag HRP and is secreted completely with the antibody, without any heterogeneous reaction. Unlike existing conventional CD34 antibodies that use heterogeneous chemical conjugation of HRP, the binding is tighter, which greatly reduces batch-to-batch variability during use.

[0018] ②HRP-CD34 antibody is a label-free secondary antibody, which simplifies the experimental procedure and improves detection efficiency. Attached Figure Description

[0019] Figure 1 Immunohistochemical results of paraffin sections of CD34Mab001-CD34Mab005, human liver tissue, CD34, under a 10x microscope. Among them, a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, c is CD34Mab001, d is CD34Mab002, e is CD34Mab004, and f is CD34Mab003.

[0020] Figure 2 The results of the immunohistochemical experiment of paraffin sections of CD34Mab005 and control antibody-human B lymphoma tissue-CD34 under 10x magnification are shown. a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is the blank control with antibody dilution solution instead of antibody.

[0021] Figure 3 Comparison of rapid immunohistochemical results of CD34Mab005 in paraffin sections of kidney tissue under CD34-10x magnification. a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is the blank control with antibody dilution solution instead of antibody.

[0022] Figure 4 Comparison of rapid immunohistochemical results of CD34Mab005 in paraffin sections of tonsil tissue under CD34-10x magnification; a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is the blank control using antibody dilution solution instead of antibody.

[0023] Figure 5 Comparison of rapid immunohistochemical results of CD34Mab005 under a CD34-10x microscope in paraffin sections of melanoma tissue; a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is the blank control using antibody dilution solution instead of antibody.

[0024] Figure 6 Comparison of CD34Mab005 rapid immunohistochemical results for frozen gastric tissue sections under CD34-10x magnification; a) CD34Mab005, b) blank control using antibody dilution buffer instead of antibody. Detailed Implementation

[0025] Example 1: Construction and validation of a transgenic mouse platform containing HRP-tagged antibodies

[0026] The construction and verification method of this embodiment refers to the invention patent with patent number ZL2023 1 1134056.1 applied for by Nanjing Fuxiao Biotechnology Co., Ltd. For the specific construction and verification method, please refer to the invention patent with patent number ZL 2023 1 1134056.1.

[0027] Example 2: Designing and screening immunogens to obtain HRP-CD34 antibody sequences

[0028] The amino acid sequence of human CD34 was obtained from NCBI. The human CD34 protein contains 595 amino acids. Uniprot was used to analyze the domains of the CD34 protein online, and the antigen index was analyzed online using http: / / www.cbs.dtu.dk / services. Based on the above analysis of the UniProt: P28906 protein structure of CD34, several immunogen design schemes were selected. Scheme ① expresses the extracellular 33-290aa sequence and performs recombinant expression as an immunogen for experiments, named Immunogen 1, with its amino acid sequence shown in SEQ ID NO: 1 and its nucleotide sequence shown in SEQ ID NO: 2; Scheme ② expresses the intracellular 312-385aa sequence, synthesizes a polypeptide, and conjugates it with BSA and OVA vectors for experiments, named Immunogen 2, with its amino acid sequence shown in SEQ ID NO: 3 and its nucleotide sequence shown in SEQ ID NO: 4; Scheme ③ tandemly expresses the extracellular 33-290aa and intracellular 312-385aa sequences for experiments, named Immunogen 3, with its amino acid sequence shown in SEQ ID NO: 5 and its nucleotide sequence shown in SEQ ID NO: 6.

[0029] Taking the mouse model of Scheme 1 established in Example 1 as an example, the specific operation steps for immunization, cell fusion, hybridoma screening, establishment of stable cell lines, and sequencing are as follows:

[0030] a. Immunized animals

[0031] 1) Select 4 mice in each group, mark and number the mice, collect 30-50ul of blood from the tail vein, and collect the pre-immune serum by centrifugation after coagulation;

[0032] 2) First immunization: Calculate the required antigen volume based on an immunization dose of 100ug per mouse, emulsify the antigen using an equal volume of Freund's complete adjuvant, and administer immunization via subcutaneous injection at multiple sites;

[0033] 3) Secondary immunization (1-week interval): Calculate the required antigen volume based on an immunization dose of 50ug per mouse, emulsify the antigen using an equal volume of Freund's incomplete adjuvant, and administer immunization via subcutaneous injection at multiple sites.

[0034] 4) Three immunizations (1 week apart): Calculate the required antigen volume based on an immunization dose of 50ug per mouse, emulsify the antigen using an equal volume of Freund's incomplete adjuvant, and administer immunization via subcutaneous injection at multiple sites.

[0035] 5) Serum collection: One week after the third immunization, 30-50 μL of blood was collected from the tail vein, and the serum was collected by centrifugation after coagulation.

[0036] 6) ELISA detection of serum titer: The initial dilution of mouse serum was 1:2000, followed by 3-fold serial dilution. The blank control group was only added with antibody diluent. The serum titer was detected by ELISA, and the OD450 reading was measured by the microplate reader. Fusion was only performed after the titer reached 1:54000.

[0037] b. Cell fusion

[0038] Mice with the highest ELISA titers were selected and boosted with an intraperitoneal injection of 50 μg / mouse three days prior to fusion. Spleens were harvested three days later for fusion. Mice were euthanized by exsanguination, and the spleens were aseptically removed, crushed, and ground in a petri dish to collect spleen cells. Pre-prepared syngeneic myeloma cells SP2 / 0 were mixed with mouse spleen cells at a ratio of 1:5, and polyethylene glycol (PEG) was added to induce fusion and form hybridoma cells. Hybridoma cells were selectively cultured in HAT medium, with a complete medium change performed seven days after fusion.

[0039] c. Hybridoma screening

[0040] The ELISA method was used for detection: the coated antigen was 1 μg / ml. Eight days after fusion, the cell culture supernatant was aspirated for ELISA detection. Wells with readings greater than 0.5 were recorded, labeled, and half of the medium was replaced. On the second day, the positive wells that had been replenished with medium were retested and specific positive tests were performed. The clone number with stable titer was recorded.

[0041] d. Establishment of stable cell lines:

[0042] From the previous step, select specific positive cell wells for subcloning using a limiting dilution method with complete culture medium, and serially dilute in 96-well plates. After 7 days, observe under a microscope, mark the single-clone wells, and perform ELISA testing the next day. Discard clones that turn negative. For positive clones, select wells with vigorous growth and perform a single culture booster. Then, select specific positive cell wells from the previous step for subcloning again using a limiting dilution method with complete culture medium, and serially dilute in 96-well plates. After 7 days, observe under a microscope, mark the single-clone wells, and perform ELISA testing the next day. Select vigorous single-cell clusters for expansion culture and cryopreservation.

[0043] The mice in Example 1 were immunized three times with different immunogen designs. It was found that the animal response titers varied greatly with different CD34 immunogen designs. The titer test results are shown in Table 1.

[0044] Table 1. Test results of different immunogenic titer regimens

[0045]

[0046] Based on the above test results, it can be seen that scheme ③ has the highest immunogenicity. Scheme ③ was selected for subsequent fusion, while the other two schemes were discarded. Scheme ③, after fusion, screening, and establishment, yielded 5 antibodies with relatively high signals, named CD34Mab001-CD34Mab005. These were tested using tissue samples. One gastrointestinal stromal tumor tissue sample and one liver tissue sample were selected. Both samples showed positive expression using the CD34 antibody reagent (product number: MAB-1076) from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Protein was extracted from the tissue samples, diluted, and coated. The samples were incubated at 37°C for 2 hours, and then 100 μL of antibodies CD34Mab001-CD34Mab005 were added to each well. After incubation at 37°C for 1 hour, the plates were washed, and the incubation was terminated promptly after adding chromogenic reagent. The OD450 reading was measured using a microplate reader. The results showed that the prepared CD34 antibody Mab005 had the best affinity and high specificity. Other antibodies showed poor affinity and non-specific reactions, as shown in Table 2.

[0047] Table 2. Immunohistochemical test results of immunogen screening antibodies in scheme ③

[0048]

[0049] To further verify the efficacy of CD34Mab001-CD34Mab005 antibodies in immunohistochemical experiments, we selected CD34 antibody (product number: MAB-1076) from Fuzhou Maixin Biotechnology Co., Ltd., and paraffin-embedded liver tissue sections from the same patient for preliminary verification. The immunohistochemical experimental procedure for the CD34 antibody from Fuzhou Maixin Biotechnology Co., Ltd. was as follows: after dewaxing and hydration of the paraffin sections, antigen retrieval was performed. After retrieval, the sections were allowed to cool naturally to room temperature. The sections were then placed in a humidified chamber with blocking buffer added. After blocking at room temperature for 20 minutes, an appropriately diluted concentration of CD34 antibody was added, and the sections were incubated at 37°C for 2 hours. Wash four times with xPBS for 5 minutes each time. Add secondary antibody at an appropriate dilution and incubate at 37°C for 1 hour. Wash four times with 1xPBS for 5 minutes each time. Add an appropriate amount of freshly prepared DAB chromogenic solution and develop at room temperature in the dark for 3-10 minutes. Observe the staining results under a microscope. After development, rinse the sections with tap water to stop the development. Counterstain the sections in hematoxylin for about 3 minutes. Wash three times with tap water. Differentiate in differentiation solution for 15 seconds. Wash three times with tap water. Re-blue in ammonia water for 30 seconds. Wash three times with tap water. Dehydrate with a series of alcohols in sequence. Clear with xylene. Mount with neutral resin mounting medium. Image and photograph the sections using an optical microscope. The antibody reaction steps obtained in this embodiment were as follows: after dewaxing and hydration of paraffin sections, antigen retrieval was performed. After retrieval, the sections were naturally cooled to room temperature, removed, placed in a humidified chamber, and blocking buffer was added. After blocking at room temperature for 1 minute, CD34 antibody of appropriate dilution was added, and the sections were incubated at 37°C for 3 minutes. The sections were washed three times with 1xPBS for 30 seconds each time. The subsequent color development and counterstaining steps were the same as the CD34 antibody immunohistochemical experiment procedure of Fuzhou Maixin Biotechnology. The results are as follows: Figure 1 As shown.

[0050] pass Figure 1 As can be seen, under the same dilution concentration and reaction conditions, the CD34Mab005 antibody obtained in the embodiments of the present invention exhibits good affinity and specificity in immunohistochemical experiments. The images clearly show that CD34Mab005 can effectively detect CD34 protein in the paraffin-embedded liver tissue sections, while other antibody strains show weaker detection signals and exhibit non-specific reactions. The CD34Mab003 antibody shows the weakest detection signal, essentially failing to detect CD34 protein in the paraffin-embedded liver tissue sections.

[0051] In summary, the CD34 antibody Mab005 obtained through immunization is the optimal antibody. Sequencing of this antibody cell line yielded the HRP-CD34 antibody sequence. The amino acid sequence of the HRP-CD34 antibody heavy chain variable region is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of the HRP-CD34 antibody light chain variable region is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0052] The amino acid sequence of the variable region of the light chain of the HRP-CD34 antibody contains a signal protein tag, horseradish peroxidase (HRP), inserted at the N-terminus of the light chain. The amino acid sequence of the variable region of the CD34 antibody light chain is shown in SEQ ID NO:11.

[0053] Example 3 Recombinant expression of HRP-CD34 antibody

[0054] The recombinant HRP-CD34 antibody was successfully fused and expressed through plasmid optimization and screening. The recombinant antibody exhibits HRP signaling protein in its light chain structure and possesses HRP signaling.

[0055] a. Obtaining recombinant antibody genes

[0056] The heavy chain variable region gene VH (sequence shown in SEQ ID NO:8) of the obtained HRP-CD34 antibody sequence was used to construct the heavy chain sequence VH-CD34FX of the recombinant HRP-CD34 antibody. Similarly, the light chain variable region gene VL (sequence shown in SEQ ID NO:10) was used to construct the light chain sequence VL-CD34FX of the recombinant HRP-CD34 antibody. Using pBI-CMV1 as a vector, plasmids carrying the recombinant genes were obtained as follows: 34pBI-CMV1-34VL (light chain) and pBI-CMV1-34VH (heavy chain). The heavy chain sequence VH-CD34FX and the light chain sequence VL-CD34FX were both synthesized by Anhui General Biotechnology Co., Ltd. The specific operation is as follows: 1) The plasmids pBI-CMV1-VL (light chain) and pBI-CMV1-VH (heavy chain) containing the heavy chain sequence and light chain sequence were transformed into competent cells by chemical transformation. After culturing on low-salt LB solid medium for 14 h, several single colonies were picked and cultured overnight in low-salt LB liquid medium. At the same time, colony PCR identification was performed using sequencing primers, and 1% agarose gel electrophoresis was used to confirm that the target gene was successfully inserted into the eukaryotic expression vector. 2) The purified plasmid was double-digested with enzymes. After digestion, 1 μL of DpnI was added to the reaction system, and the reaction was carried out at 37℃ for 30 minutes to digest the template DNA. 3) The digestion products were analyzed by 1% agarose gel electrophoresis. The target fragment was excised and extracted using a PCR product kit (FastPure GEL DNA DC301-01 Vazyme) to recover the target fragment and the pBI-CMV1 vector fragment. 4) Ligation reaction was performed. The ligation reaction system consisted of approximately 0.1 pmol of the target fragment, approximately 0.01 pmol of the pBI-CMV1 vector DNA fragment, 1 μL of T4 DNA Ligase buffer (NEB), 1 μL of T4 DNA Ligase (NEB), and deionized water was added to make up to 10 μL. Ligation was carried out overnight at 16℃. 3 μL of the reaction product was transformed into 100 μL of competent DH5α (Vazyme) cells.

[0057] b. Screening to obtain recombinant plasmids

[0058] Positive clones were selected, cultured at 37°C for 12 hours, and then plasmids were extracted using a plasmid miniprep kit (Qigen). The extracted plasmids were then sent to Shanghai Sangon Biotech for sequencing and identification.

[0059] c. Preparation of recombinant HRP-CD34 antibody

[0060] Positive clones with correct sequencing were cultured in large quantities, and plasmids were extracted according to the instructions of Tiangen Biotech (Beijing) Co., Ltd. (DP117). These were then co-transfected into healthy CHO cells. After 2 days, the culture medium was replaced with fresh medium and cultured for another 5 days. The cells were then centrifuged, and the culture supernatant was collected. The supernatant was filtered through a 0.45 μM filter tube (Millipore) and then purified using a protein A agarose gel (Changzhou Tiandi Renhe Biotechnology Co., Ltd.).

[0061] d. SDS-PAGE electrophoresis detection of HRP-CD34 antibody

[0062] Electrophoresis conditions: 5% stacking gel at 80V, 12% separating gel at 120V. Electrophoresis results showed that the constructed plasmid yielded an antibody heavy chain of 50 kDa and a light chain of 70 kDa, indicating that the HRP-CD34 antibody was successfully prepared and that the light chain contained the HRP protein structure.

[0063] e. Further validation: An immunohistochemical experiment was designed to verify the efficacy of the generated HRP-CD34 antibody. CD34 antibody (product number: MAB-1076) from Fuzhou Maixin Biotechnology Co., Ltd., and paraffin tissue sections from the same B-cell lymphoma patient were selected for preliminary validation. The immunohistochemical procedure for the CD34 antibody from Fuzhou Maixin Biotechnology was the same as above. The reaction procedure for the generated HRP-CD34 antibody was as follows: After dewaxing and hydration of the paraffin sections, antigen retrieval was performed. After retrieval, the sections were allowed to cool naturally to room temperature. The sections were then placed in a humidified chamber with blocking buffer added. After blocking at room temperature for 1 minute, an appropriately diluted concentration of HRP-CD34 antibody was added, and the sections were incubated at 37°C for 15 minutes. The sections were washed three times with 1xPBS for 30 seconds each time. Subsequent staining and counterstaining procedures were the same as those for the CD34 antibody immunohistochemical experiment from Beijing Zhongshan Jinqiao. The results are as follows: Figure 2 As shown.

[0064] The results above show that the obtained antibody has an HRP signal, which is consistent with the results of SDS-PAGE electrophoresis.

[0065] Application of the HRP-CD34 antibody prepared in Example 4 in immunohistochemical experiments

[0066] After dewaxing, hydrating, and repairing paraffin sections of kidney tissue, tonsil tissue, and melanoma using conventional methods, immunohistochemical experiments were conducted using the HRP-CD34 antibody prepared by recombinant synthesis in Example 3 and the CD34 antibody from Fuzhou Maixin Biotechnology Co., Ltd. The immunohistochemical experimental procedure for the CD34 antibody from Fuzhou Maixin Biotechnology Co., Ltd. was the same as in Example 2.

[0067] The immunohistochemical procedure for HRP-CD34 antibody was as follows: Block with blocking solution at room temperature for 1 minute, remove the blocking solution, add the HRP-CD34 antibody working solution obtained above, incubate at 37°C for 15 minutes, wash three times with 1xPBS for 30 seconds each time, then add freshly prepared DAB chromogenic solution and incubate at room temperature in the dark for 2-3 minutes. Observe the chromogenic results under an optical microscope. After chromogenic development, rinse the slides with tap water to stop the chromogenic process, counterstain with hematoxylin for 3 minutes, rinse three times with tap water, treat with differentiation solution for 15 seconds, rinse three times with tap water, perform ammonia blueing for 30 seconds, rinse three times with tap water, dehydrate, clear, mount, photograph and save the results. See below for details. Figure 3 , Figure 4 , Figure 5 During the experiment, a blank control group was set up (incubated with 1xPBS instead of HRP-CD34 antibody).

[0068] The above experimental results show that using the HRP-CD34 antibody prepared in this invention for immunohistochemical experiments can greatly shorten the immunohistochemical detection time (reducing the addition of secondary antibody and incubation reaction time) and has high detection accuracy (no significant difference from the CD34 antibody detection results of Beijing Zhongshan Jinqiao), making it possible to quickly complete the immunohistochemical experiment of paraffin sections.

[0069] Application of the HRP-CD34 antibody prepared in Example 5 in frozen immunohistochemistry experiments

[0070] To further verify the clinical application performance of the HRP-CD34 antibody prepared in this invention, frozen sections of gastric tissue were selected for verification. After fixing the frozen sections with alcohol for 1 minute, blocking buffer was added and blocked at room temperature for 1 minute. The blocking buffer was then removed, and the HRP-CD34 antibody working solution prepared above was added directly. The sections were incubated at room temperature for 5 minutes, washed three times with 1xPBS for 30 seconds each time, and then freshly prepared DAB chromogenic solution was added. The sections were then incubated at room temperature in the dark for 2 minutes. The staining results were observed under an optical microscope. Subsequent procedures included counterstaining, blue staining, dehydration, clearing, mounting, and photographing. The experimental results are shown below. Figure 6 During the experiment, a blank control group was set up (incubated with 1xPBS instead of HRP-CD34 antibody).

[0071] The above experimental results show that using the HRP-CD34 antibody prepared in this invention for immunohistochemical experiments can greatly shorten the immunohistochemical detection time without affecting the experimental results, making it possible to complete the immunohistochemical experiment of frozen sections in about 10-15 minutes.

[0072] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A CD34 biofusion enzyme antibody with a signal protein tag, characterized in that, The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

7.

2. The nucleotide sequence encoding the CD34 biofusion enzyme antibody with a signal protein tag as described in claim 1.

3. An expression carrier, characterized in that, The expression vector contains the nucleotide sequence of claim 2.

4. A recombinant host cell containing the expression vector of claim 3.

5. A method for preparing the CD34 biofusion enzyme antibody with a signal protein tag as described in claim 1, characterized in that, The method includes: 1) Under suitable conditions, the recombinant host cells of claim 4 are cultured to express antibodies; 2) Then the antibodies are purified and collected from the host cells.

6. The use of the CD34 biofusion enzyme antibody with signal protein tag as described in claim 1 in the preparation of a CD34 target immunohistochemical detection kit.

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