CD34 biological fusion enzyme antibody with signal protein tag and application

Abstract

The invention discloses a CD34 biological fusion enzyme antibody with a signal protein tag and application, the amino acid sequence of a light chain variable region of an anti-human CD34 antibody is shown as SEQ ID NO: 11, the amino acid sequence of a heavy chain variable region of the anti-human CD34 antibody is shown as SEQ ID NO: 7, the signal protein tag HRP is inserted into the N end of the light chain of the anti-human CD34 antibody, and the amino acid sequence of the light chain variable region of the obtained HRP-CD34 antibody is shown as SEQ ID NO: 9. In addition, the invention also provides a preparation method of the HRP-CD34 antibody, the HRP-CD34 antibody is a molecule completely secreted along with the antibody, has no heterogeneity, is good in sensitivity, high in specificity and stable in performance, and does not need to add a secondary antibody during detection, so that the cost is saved, and the efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological disease detection, and in particular to a CD34 biofusion enzyme antibody with a signal protein tag and an application thereof. Background Art

[0002] CD34 is a single-chain transmembrane glycoprotein with a relative molecular mass of 110,000 encoded by a gene located at 1q32. It can be expressed in normal endothelial cells, spleen marginal zone cells, and dendritic interstitial cells surrounding blood vessels, nerves, muscle bundles, skin appendages, and mammary lobular matrix. CD34 is positively expressed in vascular tumors. CD34 can also be expressed in the following tissues: immature leukemia (including AML, ALL, and granulocytic sarcoma), lymphoblastic lymphoma, transformed myelodysplasia, smooth muscle cells and tumors derived from them, gastrointestinal stromal tumors, neurothecomas, lipomas, epithelioid sarcomas, etc. Benign and malignant fibrous histiocytomas and fibrosarcomas are rarely positive, and sarcomatoid mesothelioma, synovial sarcoma, and fibromatosis do not express CD34. CD34 is mainly used to determine whether there is endothelial cell differentiation. It is used in combination with CD117 to identify gastrointestinal stromal tumors, 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, the commonly used CD34 immunohistochemistry kits currently have the following disadvantages: 1. The required staining time is relatively long, which cannot meet the clinical demand for rapid intraoperative pathological diagnosis, so its application is limited. It can only be used for pathological auxiliary diagnosis of conventional paraffin sections, and cannot be used for pathological auxiliary diagnosis of intraoperative frozen sections. 2. Signal detection is achieved by chemically coupling signal enzymes such as HRP to CD34 antibodies. The coupling efficiency is difficult to control, resulting in large differences in antibody staining results between different batches; in addition, the coupling yield is very low, resulting in a great waste of antibodies. Therefore, there is an urgent need for an immunohistochemistry kit for CD34 target detection with a short staining time and accurate and stable staining results. Summary of the invention

[0004] In view of the above problems, the present invention provides an HRP-CD34 antibody, which is a CD34 bio-fusion enzyme antibody with a signal protein tag HRP, and the signal protein tag HRP is completely secreted together 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 a sequence similarity of more than 80% to SEQ ID NO. 11; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7 or a sequence having a sequence similarity of more than 80% to SEQ ID NO. 7.

[0006] Express the nucleotide sequence of the anti-human CD34 antibody.

[0007] A CD34 biofusion enzyme antibody with a signal protein tag, wherein the antibody is prepared by inserting a signal protein tag into the N-terminus of the light chain of the above-mentioned 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 a signal protein tag is shown in SEQ ID NO:9.

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

[0010] An expression vector comprising the above-mentioned nucleotide sequence.

[0011] A recombinant host cell containing the above expression vector.

[0012] A method for preparing the CD34 biofusion enzyme antibody with a signal protein tag comprises: 1) culturing the recombinant host cell to express the antibody under appropriate conditions; 2) The antibodies are then purified and collected from the host cells.

[0013] The application of the anti-human CD34 antibody and the CD34 biofusion enzyme antibody with a signal protein tag in the preparation of a CD34 target immunohistochemistry detection kit. Beneficial Effects

[0014] Different from the prior art, the advantages of the present invention are: ① HRP-CD34 antibody carries its own signal protein tag HRP, which is completely secreted with the antibody without any heterogeneous reaction. Unlike the existing conventional CD34 antibody that is coupled to HRP through heterogeneous chemistry, the connection is tighter, and the batch-to-batch difference is greatly reduced during use; ②HRP-CD34 antibody does not require labeled secondary antibody, which simplifies the experimental steps and improves detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The immunohistochemical results of CD34Mab001-CD34Mab005 paraffin sections-human liver tissue-CD34-10 times microscope, where a is MAB-1076 of Maixin Biotechnology, b is CD34Mab005, c is CD34Mab001, d is CD34Mab002, e is CD34Mab004, and f is CD34Mab003; Figure 2 The results of the immunohistochemistry experiment of CD34Mab005 and control antibody paraffin sections - human B lymphoma tissue - CD34 - 10 times microscope, a is MAB-1076 of Maixin Bio, b is CD34Mab005, and c is the blank control with antibody diluent instead of antibody; Figure 3 This is a comparison of the results of CD34-10x microscope and CD34Mab005 rapid immunohistochemistry of paraffin sections of kidney tissue. a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is a blank control using antibody diluent instead of antibody. Figure 4 This is a comparison of the results of CD34-10x microscope and CD34Mab005 rapid immunohistochemistry of tonsil tissue paraffin sections; a is MAB-1076 from Maixin Biotechnology, b is CD34Mab005, and c is a blank control using antibody diluent instead of antibody; Figure 5 This is a comparison of CD34-10x microscope and CD34Mab005 rapid immunohistochemistry results of paraffin sections of melanoma tissue; a is MAB-1076 of Maixin Bio, b is CD34Mab005, and c is a blank control using antibody diluent instead of antibody; Figure 6 Comparison of CD34-10x microscope and CD34Mab005 rapid immunohistochemistry results of gastric tissue frozen sections; a is CD34Mab005, b is blank control with antibody diluent instead of antibody. DETAILED DESCRIPTION

[0016] Example 1: Construction and verification of a transgenic mouse platform producing antibodies containing an HRP tag The construction and verification methods of this embodiment refer to the invention patent with patent number ZL2023 1 1134056.1 applied for by Nanjing Fuxiao Biotechnology Co., Ltd., and the specific construction and verification methods are shown in the invention patent with patent number ZL 2023 1 1134056.1.

[0017] Example 2: Design and screening of immunogens to obtain HRP-CD34 antibody sequences The amino acid sequence of human CD34 was obtained from NCBI. The human CD34 protein contains 595 amino acids. The domain structure of the CD34 protein was analyzed online using Uniprot, and the antigenic index was analyzed online using http: / / www.cbs.dtu.dk / services. According to the above analysis of CD34 UniProt: P28906 protein structure, several schemes were selected for immunogen design. Scheme ① The expression sequence is extracellular 33-290aa, which is recombinantly expressed as an immunogen for experiments and named as immunogen one. Its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 2; Scheme ② Intracellular 312-385aa, a synthetic polypeptide is coupled to BSA and OVA vectors for experiments and named as immunogen two. Its amino acid sequence is shown in SEQ ID NO: 3, and its nucleotide sequence is shown in SEQ ID NO: 4; Scheme ③ The extracellular 33-290aa and intracellular 312-385aa are expressed in tandem and then experimented. It is named as immunogen three. Its amino acid sequence is shown in SEQ ID NO: 5, and its nucleotide sequence is shown in SEQ ID NO: 6.

[0018] Taking the mouse model of Scheme 1 constructed in Example 1 as an example, immunization, cell fusion, hybridoma screening, establishment of stable strains, and sequencing were performed. The specific operation steps are as follows: a. Immunization of animals 1) Four mice were selected from each group, and the mice were marked and numbered. 30-50ul of blood was collected from the tail vein, and the pre-immune serum was collected by centrifugation after coagulation; 2) First immunization: Calculate the required antigen volume based on the immunization dose of 100ug per mouse, use an equal volume of Freund's complete adjuvant to emulsify the antigen, and immunize by subcutaneous injection at multiple points; 3) Second immunization (1 week apart): Calculate the required antigen volume based on the immunization dose of 50 ug per mouse, use an equal volume of Freund's incomplete adjuvant to emulsify the antigen, and immunize by subcutaneous injection at multiple points; 4) Three immunizations (1 week apart): Calculate the required antigen volume based on the immunization dose of 50 ug per mouse, use an equal volume of Freund's incomplete adjuvant to emulsify the antigen, and immunize with multiple subcutaneous injections; 5) Serum collection: One week after the third immunization, collect 30-50ul of blood from the tail vein, and collect serum by centrifugation after blood coagulation; 6) ELISA test for serum titer: The initial dilution of mouse serum was 1:2000, and it was diluted 3 times. Only antibody diluent was added to the blank control group. ELISA was used to test the serum titer. The OD450 reading was measured by an enzyme marker. Fusion was performed only when the titer reached 1:54000.

[0019] b. Cell fusion Select the mouse with the highest ELISA titer, and give the mouse a booster immunization 3 days before fusion, 50ug / mouse, intraperitoneal injection, and take the spleen for fusion 3 days later. Kill the mouse by enucleation and bleeding, remove the spleen aseptically, squeeze and grind in a plate, and collect spleen cells. Mix the prepared syngeneic myeloma cells SP2 / 0 with mouse spleen cells in a certain ratio (1:5), add the fusion promoter polyethylene glycol, and fuse the cells to form hybridoma cells. Use HAT medium for selective culture of hybridoma cells, and completely change the medium once 7 days after fusion.

[0020] c. Hybridoma Screening ELISA method was used for detection: coated antigen with a concentration of 1ug / ml, and cell culture supernatant was aspirated for ELISA detection 8 days after fusion. Wells with readings greater than 0.5 were recorded, marked, and half-fluid replacement was performed. On the second day, positive wells that were replenished were retested and specifically tested, and the clone numbers with stable titers were recorded.

[0021] d. Establishment of stable cell lines: Select specific positive cell wells from the previous step for subclone screening, limit dilution method, complete medium, and dilute in a 96-well plate in a gradient manner; observe under a microscope after 7 days, mark the monoclonal wells, perform ELISA test the next day, discard the clone numbers that turn negative, and for positive clones, select wells with vigorous growth for a rehydration; select specific positive cell wells from the previous step for subclone screening, limit dilution method, complete medium, and dilute in a 96-well plate in a gradient manner; observe under a microscope after 7 days, mark the monoclonal wells, perform ELISA test the next day, select single cell clusters with vigorous growth for expansion culture, and freeze for seed preservation.

[0022] The mice of Example 1 were immunized three times with different immunogens. It was found that the animal response titers varied greatly with different CD34 immunogen designs. The titer test results are shown in Table 1.

[0023] Table 1 Test results of different immunogen titer schemes

[0024] According to the above test results, it can be seen that the immunogenic titer of scheme ③ is the highest, and this scheme is selected for subsequent fusion. The other two groups of schemes are discarded. After fusion, screening and strain construction, 5 antibody strains with relatively high signals are obtained in scheme ③, named CD34Mab001-CD34Mab005, which are tested with tissue samples respectively. One gastrointestinal stromal tumor tissue sample and one liver tissue sample are selected. The above samples are positively expressed by CD34 antibody (product number: MAB-1076) reagent of Beijing Zhongshan Jinqiao Biological Co., Ltd. The protein in the tissue samples is extracted, coated after dilution, incubated at 37℃ for 2 hours, and then added with antibodies CD34Mab001-CD34Mab005, 100ul is added to each well, and the plate is washed after 1 hour at 37℃. The color development solution is added and the color is stopped in time. The OD450 reading is measured by an enzyme marker. The results show that the prepared CD34 antibody Mab005 has the best affinity effect and high specificity. The affinity of other antibodies was poor and there were non-specific reactions. The experimental results are shown in Table 2.

[0025] Table 2 Results of immunohistochemistry test of immunogen screening antibodies in scheme ③

[0026] To further verify the effects of CD34Mab001-CD34Mab005 in immunohistochemical experiments, CD34 antibody (product number: MAB-1076) from Fuzhou Maixin Biotechnology Co., Ltd. and paraffin tissue sections of the liver of the same patient were selected for preliminary verification. The CD34 antibody immunohistochemical experiment operation steps of Fuzhou Maixin Biotechnology Co., Ltd. are as follows: paraffin sections are dewaxed and hydrated, and antigen repair is performed. After repair, they are naturally cooled to room temperature, and the sections are taken out and placed in a wet box and blocked with blocking solution. After blocking at room temperature for 20 minutes, CD34 antibody of appropriate dilution concentration is added, and incubated at 37°C for 2 hours. Wash with xPBS 4 times for 5 minutes each time, add secondary antibody of appropriate dilution concentration, incubate at 37℃ for 1 hour, wash with 1xPBS 4 times for 5 minutes each time, add appropriate amount of freshly prepared DAB color developing solution, color at room temperature in the dark for 3-10 minutes, observe the staining results under a microscope, rinse the sections with tap water after color development to terminate color development, put the sections in hematoxylin for counterstaining for about 3 minutes, wash with tap water 3 times, put them in differentiation solution for differentiation for 15 seconds, wash with tap water 3 times, turn blue in ammonia water for 30 seconds, wash with tap water 3 times, dehydrate with gradient alcohol in sequence, make transparent with xylene, seal with neutral gum sealing medium, and image and photograph the sections with an optical microscope. The antibody reaction steps obtained in this example are: after the paraffin sections are dewaxed and hydrated, antigen repair is performed, and after repair, the sections are naturally cooled to room temperature, the sections are taken out and placed in a wet box and a blocking solution is added, and after blocking at room temperature for 1 minute, a CD34 antibody with an appropriate dilution concentration is added, incubated at 37°C for 3 minutes, and washed 3 times with 1xPBS, each for 30 seconds. The subsequent color development and counterstaining steps are the same as the CD34 antibody immunohistochemistry experimental operation steps of Fuzhou Maixin Biology. The results are shown in Figure 1 shown.

[0027] pass Figure 1 It can be seen that under the same dilution concentration and reaction conditions, the CD34Mab005 antibody obtained in the embodiment of the present invention has better affinity and specificity in the immunohistochemistry experiment. It can be clearly seen from the picture that CD34Mab005 can well detect the CD34 protein in the paraffin section of the liver tissue, while the detection signals of other antibody strains are relatively weak and have non-specific reactions. The detection signal of CD34Mab003 antibody is the weakest, and CD34 protein in the paraffin section of the liver tissue is basically not detected.

[0028] In summary, the CD34 antibody Mab005 obtained by immunization is the optimal antibody. The antibody cell line was sequenced to obtain the HRP-CD34 antibody sequence. After testing, the amino acid sequence of the heavy chain variable region of the HRP-CD34 antibody is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:8; the amino acid sequence of the light chain variable region of the HRP-CD34 antibody is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:10.

[0029] In the amino acid sequence of the light chain variable region of the HRP-CD34 antibody, a signal protein tag horseradish peroxidase HRP is inserted at the N-terminus of the light chain. The amino acid sequence of the light chain variable region of the CD34 antibody is shown in SEQ ID NO:11.

[0030] Example 3 Recombinant expression of HRP-CD34 antibody The recombinantly expressed HRP-CD34 antibody was successfully fused and expressed through plasmid optimization screening, showing HRP signal protein on the light chain structure and having HRP signal.

[0031] a. Acquisition of recombinant antibody genes The heavy chain variable region gene VH (sequence as 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, and the light chain variable region gene VL (sequence as 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, the plasmids carrying the recombinant genes were obtained, namely 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 operations were 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, cultured on low-salt LB solid culture medium for 14 hours, and several single colonies were picked and cultured overnight in low-salt LB liquid culture medium. At the same time, colony PCR identification was performed using sequencing primers, and 1% agarose gel electrophoresis was used to detect, proving that the target gene was successfully inserted into the eukaryotic expression vector. 2) The purified plasmid was double-digested. After the digestion was completed, 1μL 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 product was analyzed by 1% agarose gel electrophoresis, and the target fragment was cut out. The target fragment and pBI-CMV1 vector fragment were extracted and recovered by PCR product kit (FastPure GEL DNA DC301-01 Vazyme); 4) The ligation reaction was performed; the ligation reaction system was: about 0.1pmol target fragment, about 0.01pmol pBI-CMV1 vector DNA fragment, 1μL T4 DNA Ligase buffer (NEB), 1μL T4 DNA Ligase (NEB), deionized water was added to make up to 10μL system, and the ligation was carried out at 16℃ overnight. Take 3μL of the reaction product and transform it into 100μL competent cells DH5a (Vazyme); b. Screening to obtain recombinant plasmid Positive clones were picked and cultured at 37°C for 12 h. The plasmids were extracted using a plasmid extraction kit (Qigen) and sent to Shanghai Biotechnology for sequencing and identification.

[0032] c. Preparation of recombinant HRP-CD34 antibody The positive clones with correct sequencing were shaken in large quantities, and the plasmids were extracted according to the instructions (DP117 of Tiangen Biochemical Technology (Beijing) Co., Ltd.), co-transfected into CHO cells in good condition, and fresh medium was replaced after 2 days to continue culturing for 5 days, centrifuged, and the culture supernatant was collected. It was filtered using a 0.45uM pore filter tube (Millipore), and then purified using protein A agarose gel (Changzhou Tiandi Renhe Biological Co., Ltd.).

[0033] d. SDS-PAGE electrophoresis detection of HRP-CD34 antibody Electrophoresis conditions: 5% concentrated gel 80V voltage, 12% separation gel 120V voltage. The electrophoresis results showed that the antibody heavy chain size obtained by constructing the plasmid was 50KDa, and the light chain size was 70KDa, indicating that the HRP-CD34 antibody was successfully prepared and there was an HRP protein structure in the antibody light chain part.

[0034] e. For further verification, an immunohistochemistry experiment was designed to verify the effect of the generated HRP-CD34 antibody. The CD34 antibody (product number: MAB-1076) of Fuzhou Maixin Biotechnology Co., Ltd. and the paraffin tissue sections of the same B lymphoma patient were selected for preliminary verification. The operation steps of the CD34 antibody immunohistochemistry experiment of Fuzhou Maixin Biotechnology (same as above); the reaction steps of the generated HRP-CD34 antibody are as follows: the paraffin sections are dewaxed and hydrated, and then antigen repair is performed. After repair, they are naturally cooled to room temperature, and the sections are taken out and placed in a wet box and the blocking solution is added. After blocking at room temperature for 1 minute, the HRP-CD34 antibody of appropriate dilution concentration is added, incubated at 37°C for 15 minutes, and washed 3 times with 1xPBS for 30 seconds each time. The subsequent color development and counterstaining steps are the same as the CD34 antibody immunohistochemistry experiment operation steps of Beijing Zhongshan Jinqiao. The results are as follows Figure 2 shown.

[0035] From the above results, it can be seen that the obtained antibody has HRP signal, which is consistent with the SDS-PAGE electrophoresis detection result.

[0036] Example 4 Application of the prepared HRP-CD34 antibody in immunohistochemistry experiments After dewaxing, hydration and repair of paraffin sections of kidney tissue, tonsil tissue and melanoma in a conventional manner, immunohistochemistry experiments were performed using the recombinant HRP-CD34 antibody prepared in Example 3 and the CD34 antibody of Fuzhou Maixin Biotechnology Co., Ltd., where the immunohistochemistry experimental operation steps of the CD34 antibody of Fuzhou Maixin Biotechnology Co., Ltd. were the same as those of Example 2.

[0037] The immunohistochemistry experiment of HRP-CD34 antibody was performed as follows: block with blocking solution at room temperature for 1 minute, remove the blocking solution, directly add the HRP-CD34 antibody working solution obtained above, incubate at 37° for 15 minutes, wash 3 times with 1xPBS, 30 seconds each time, then add freshly prepared DAB color developing solution, color at room temperature in the dark for 2-3 minutes, observe the color development results under an optical microscope, rinse the sections with tap water after color development to terminate color development, counterstain with hematoxylin for 3 minutes, rinse 3 times with tap water, treat with differentiation solution for 15 seconds, rinse 3 times with tap water, blue with ammonia water for 30 seconds, rinse 3 times with tap water, dehydrate, make transparent, seal the sections, take pictures and keep them, and the experimental results are shown in the table. Figure 3 , Figure 4 , Figure 5 During the experiment, a blank control group was set up (1xPBS was used instead of HRP-CD34 antibody for incubation).

[0038] The above experimental results show that the use of the HRP-CD34 antibody prepared by the present invention for immunohistochemical experiments can greatly shorten the immunohistochemical detection time (reducing the addition of secondary antibodies and incubation reaction time) and has high detection accuracy (no significant difference with the CD34 antibody detection results of Beijing Zhongshan Jinqiao), making it possible to quickly complete paraffin section immunohistochemical experiments.

[0039] Example 5 Application of the prepared HRP-CD34 antibody in frozen immunohistochemistry experiments In order to further verify the clinical application performance of the HRP-CD34 antibody prepared by the invention, frozen sections of gastric tissue were selected for verification. After the frozen sections were fixed with alcohol for 1 minute, blocking solution was added and blocked at room temperature for 1 minute. The blocking solution was removed and the HRP-CD34 antibody working solution obtained above was directly added. The sections were incubated at room temperature for 5 minutes, washed 3 times with 1xPBS, each for 30 seconds, and then freshly prepared DAB color developing solution was added. The sections were color developed at room temperature in the dark for 2 minutes. The staining results were observed under an optical microscope, and then counterstained, blued, dehydrated, transparent, sealed, and photographed for preservation. The experimental results are shown in Figure 6 During the experiment, a blank control group was set up (1xPBS was used instead of HRP-CD34 antibody for incubation).

[0040] The above experimental results show that the use of the HRP-CD34 antibody prepared by the present invention in immunohistochemical experiments can greatly shorten the immunohistochemical detection time without affecting the experimental results, making it possible to complete frozen section immunohistochemical experiments in about 10-15 minutes.

[0041] It will be apparent to those skilled in the art that the 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 the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. An anti-human CD34 antibody, characterized in that The amino acid sequence of the light chain variable region of the antibody is SEQ ID NO: 11 or a sequence having a sequence similarity of more than 80% to SEQ ID NO. 11; the amino acid sequence of the heavy chain variable region is SEQ ID NO: 7 or a sequence having a sequence similarity of more than 80% to SEQ ID NO.

7.

2. A nucleotide sequence expressing the anti-human CD34 antibody according to claim 1.

3. A CD34 biofusion enzyme antibody with a signal protein tag, characterized in that: The antibody is an antibody in which a signal protein tag is inserted into the N-terminus of the light chain of the anti-human CD34 antibody of claim 1.

4. The CD34 biofusion enzyme antibody with a signal protein tag according to claim 3, characterized in that: The signal protein tag is horseradish peroxidase HRP.

5. The CD34 biofusion enzyme antibody with a signal protein tag according to claim 4, characterized in that: The amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:

9.

6. A nucleotide sequence encoding the CD34 biofusion enzyme antibody with a signal protein tag according to claim 5.

7. An expression vector, characterized in that: The expression vector contains the nucleotide sequence according to claim 2 or 6.

8. A recombinant host cell containing the expression vector according to claim 7.

9. A method for preparing the CD34 biofusion enzyme antibody with a signal protein tag according to claim 5, characterized in that: The method comprises: 1) culturing the recombinant host cell according to claim 8 to express the antibody under appropriate conditions; 2) The antibodies are then purified and collected from the host cells.

10. Use of the anti-human CD34 antibody of claim 1 and the CD34 biofusion enzyme antibody with a signal protein tag of claim 3 in the preparation of a CD34 target immunohistochemical detection kit.

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