Monoclonal antibody a5 of colp6 of marnifia vesfa and its preparation method and application

Abstract

The application discloses a monoclonal antibody A5 of Marninifia colp6, a light chain variable region of the monoclonal antibody A5 has an amino acid sequence as shown in SEQ ID NO. 1, and a heavy chain variable region has an amino acid sequence as shown in SEQ ID NO. 2; and the application also discloses a preparation method and application of the monoclonal antibody. The light chain variable region gene sequence and the amino acid sequence of the heavy chain variable region of the monoclonal antibody of Marninifia colp6 are successfully cloned, the monoclonal antibody A5 with the light chain variable region and the heavy chain variable region has the characteristics of being capable of specifically recognizing Marninifia, high specificity and high detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody A5 against *Cladosporium marneffei* colp6, its preparation method, and its application. Background Technology

[0002] *Penicillium marneffei* (PM), also known as *Basilella marneffei*, is a dimorphic fungus that causes marneffei fungal infection (PSM), a rare systemic disease caused by a deep fungal infection. *Basilella marneffei* infection is most common in southern China and Southeast Asia. While it can occur in healthy individuals, it is more prevalent in immunocompromised or immunosuppressed individuals. Studies in Vietnam have shown that PSM ranks third among opportunistic infections in AIDS patients. With the increasing number of HIV infections, reports of *Penicillium marneffei* infection are also rising annually.

[0003] Existing literature reports that *Basilaria marneffei* glycoproteins and *Aspergillus* glycoproteins have the same or similar antigenic determinants, making cross-reactivity in immunological detection easy. Furthermore, glycoproteins are easily cleared by phagocytes after binding with antibodies. However, a rapid, sensitive, and specific method for detecting *Basilaria marneffei* antigens has not yet been developed. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a colp6 monoclonal antibody A5 of *Basilella marneffei*, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0006] A monoclonal antibody A5 against *Cladosporium marneffei* colp6, wherein the protein sequence of the monoclonal antibody A5 contains a light chain variable region and a heavy chain variable region, the light chain variable region having the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region having the amino acid sequence shown in SEQ ID NO.2; the light chain variable region and the heavy chain variable region are linked by a short peptide having the amino acid sequence shown in SEQ ID NO.3.

[0007] The nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.5.

[0008] The preparation method of the above-mentioned monoclonal antibody A5 includes the following steps:

[0009] The gene for synthesizing the Colp6 protein of Marneffei was synthesized, and the recombinant Colp6 protein was expressed and purified. Using the recombinant Colp6 protein and Marneffei as immunogens, Balb / c mice were cross-immunized. Spleen cells from the immunized mice were fused with mouse myeloma NS-1 cells. Hybridoma cells secreting specific McAbs were screened by indirect ELISA. Ascites was induced in mice using hybridoma cell lines, and the antibody was purified by protein G affinity chromatography. Finally, the Colp6 protein monoclonal antibody A5 was obtained.

[0010] Furthermore, the amino acid sequence of the Marneffei Colp6 protein is shown in SEQ ID NO.6.

[0011] Furthermore, the preparation method includes the following steps:

[0012] Step S1: Synthesis of the Colp6 gene

[0013] The amino acid sequence of the Colp6 gene from *Marneffeis* was obtained from the GenBank sequence database; codon optimization of the Colp6 gene base sequence was performed based on the codon preference of *Escherichia coli*, and the codon-optimized gene sequence was synthesized.

[0014] Step S2: Expression and purification of the Colp6 gene

[0015] The Colp6 gene was ligated into the pQE 80L vector to obtain a recombinant vector. The plasmid of the recombinant vector was extracted and transformed into E. coli BL21(DE3). Expression was induced by IPTG. The bacterial cells were collected, high-pressure lysed, and the supernatant was collected after centrifugation. The protein was purified by Ni column affinity chromatography. The purified protein was further purified using a Superdex-200 column to obtain a high-purity Colp6 recombinant protein. The Colp6 recombinant protein was collected as an antigen for later use.

[0016] Step S3, mouse immunization

[0017] Purified Colp6 recombinant protein and Marneffei bacteria were used as immunogens and antigens. Three female BALB / c mice aged 6-8 weeks were immunized. Ten days after routine immunization, blood was collected from the tail vein and the antibody titers produced by the mice were detected by indirect ELISA. Three days before fusion, the recombinant antigen was injected for the last time via the tail vein at a dose of 60 μg / mouse for a booster immunization.

[0018] Step S4, Preparation of NS-1 myeloma cells

[0019] One week prior to fusion, NS-1 myeloma cells preserved in liquid nitrogen were revived and cultured in 25 cm⁻¹ medium. 2In cell culture flasks, cells were passaged in DMEM medium containing 15% fetal bovine serum for one week, and the cell concentration was adjusted to 10⁻⁶. 6 Cells / mL; during fusion, select myeloma cells in the logarithmic growth phase, discard the original culture medium in the bottle, add an appropriate amount of serum-free DMEM culture medium, gently blow the cells off, transfer them to a 50mL centrifuge tube, centrifuge at 400g for 5 minutes, wash the cells 3 times, discard the supernatant, resuspend the cell pellet in serum-free culture medium, and count them for later use.

[0020] Step S5, Preparation of immune spleen cells

[0021] One BALB / c mouse with the best immune response was euthanized by bleeding from the eye socket and disinfected by soaking in 75% alcohol for 5 minutes. The disinfected mouse was fixed in a laminar flow hood, and the spleen was removed. Adhesive and connective tissue adhering to the cells were removed with scissors. The spleen was rinsed with serum-free culture medium and transferred to a 40μm cell filter. The cells were gently ground with a syringe core, and the filter was gently rinsed with serum-free culture medium. The spleen cell suspension was collected. The cells were centrifuged at 400g for 5 minutes, washed three times, and the supernatant was discarded. The cell pellet was resuspended in serum-free DMEM culture medium and counted for later use.

[0022] Step S6, Cell Fusion

[0023] Mix NS-1 myeloma cells and immune spleen cells at a 1:10 ratio in a 50 mL centrifuge tube, centrifuge at 400 g for 10 min, aspirate the supernatant, and gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly. Slowly add 1 mL of 50% PEG solution pre-warmed to 37°C over 1 min, continuously and gently stirring the cells with a pipette for 1 min. Then add 10 mL of DMEM medium to the cell mixture, adding 1 mL dropwise over the first minute, 1 mL over the second minute, 3 mL over the third and fourth minutes, and the remaining 5 mL over the fifth minute. Incubate the cell mixture in a 37°C water bath for 15 min. Centrifuge at 400 g for 5 min, remove the supernatant, resuspend the cell pellet in 20 mL of 15% fetal bovine serum DMEM medium, and transfer to a 75 cm⁻¹ centrifuge tube. 2 Incubate the cells in a cell culture flask for 16–24 hours. Transfer the fused cell suspension to a 50 mL centrifuge tube and centrifuge at 400 g for 10 min. Remove the supernatant and resuspend the cell pellet in 2.5 mL of 15% fetal bovine serum DMEM medium. Add 22.5 mL of semi-solid medium, mix well, and pour into 3.5 cm diameter petri dishes, about 2 mL per dish. Incubate at 37 °C with 5% CO2. After ten days, visible white cell clones will appear on the surface of the culture medium in the petri dishes.

[0024] Step S7, Screening for positive hybridoma cells

[0025] Under aseptic conditions, cell clusters were aspirated from petri dishes and placed into 96-well plates for culture for 4 days. After 4 days, indirect ELISA was performed. Serum from normal mice before immunization was used as a negative control, and serum from mice after immunization was used as a positive control. A positive result was determined by an absorbance value 2.1 times greater than that of the negative control. Wells showing specific antibodies and exhibiting single-clone growth with good morphology were re-cloned. After at least 3 re-cloning, the hybridoma cells from the positive wells were transferred to 24-well plates. Once the hybridoma cells in the 24-well plates were growing well, the hybridoma cells were cryopreserved.

[0026] Step S8, Preparation and purification of monoclonal antibody A5

[0027] 0.5 mL of liquid paraffin was injected into the peritoneal cavity of 8-week-old BALB / c mice; hybridoma cells were then injected at a concentration of 1 × 10⁻⁶. 5 Inoculate at a concentration of cells / ml at a 25cm depth. 2 In a culture flask, culture until the cells reach optimal viability, then adjust the cell count to approximately 1×10⁶. 6 The ascites was inoculated into the peritoneal cavity of mice that had been injected with liquid paraffin; ascites fluid was collected 7–10 days later; the ascites fluid was diluted more than 3 times with 20 mM PBS pH 7.4, centrifuged and the supernatant was collected and purified by HiTrap protein G affinity chromatography to obtain Colp6 protein monoclonal antibody A5.

[0028] Further, in step S3, the immunization method for mice is as follows: 60 μg of the original emulsion is used per mouse, and the total volume of antigen + 1×PBS is 350 μL. This mixture is then emulsified 3500 times at 200 times / min with an equal volume of Freund's adjuvant. For the first immunization, 0.2 mL of the emulsion containing 60 μg of antigen is subcutaneously injected into the groin of each mouse using a 2 mL syringe, and the injection time and site are recorded. For the second immunization, two weeks later, 0.2 mL of the emulsion containing 60 μg of antigen is injected intraperitoneally into each mouse using a 2 mL syringe, and the injection time and site are recorded. For the third immunization, two weeks later, 0.2 mL of the emulsion containing 60 μg of antigen is injected subcutaneously into the groin of each mouse using a 2 mL syringe, and the injection time and site are recorded.

[0029] Further, in step S7, the indirect ELISA detection method is as follows: the antigen is coated onto the ELISA plate at 100 ng / well, incubated overnight at 4°C, washed three times, 200 mL of blocking buffer is added, and the plate is incubated at 37°C for 2 h, washed three times, then hybridoma cell culture supernatant is added, and the plate is incubated at 37°C for 2 h, washed three times with washing buffer, then goat anti-mouse IgG-HRP diluted 1:10000 is added, and the plate is incubated at 37°C for 1 h, washed three times, and then developed at 37°C in the dark for 10 min. After sufficient development, 2 M H2 SO4 is added to terminate the reaction, and the absorbance (OD value) is measured at a wavelength of 450 nm using an ELISA reader.

[0030] Further, in step S8, purification is performed using a Protein G affinity chromatography column. A new column is first passed through 5 mL of ultrapure water, then equilibrated with 5 mL of 0.4 M PB buffer (pH 7.0). The antibody is passed through the column slowly to ensure better binding of the antibody protein to the binding site. The column is then equilibrated again with 10 mL of 0.4 M pH 7.0 PB buffer. 5 mL of 0.1 M pH 2.7 glycine-hydrochloric acid buffer is used to remove the antibody from the binding site, and 1 M pH 8.0 Tris-HCl is added to neutralize the glycine, maintaining a neutral pH suitable for antibody preservation. The purified antibody is then used for hybridoma cell sequencing.

[0031] The present invention also protects the application of monoclonal antibody A5 against *Cladosporium marneffei* colp6, for any one of the following (a1) to (a6):

[0032] (a1) Identification of *Basilella marneffei*;

[0033] (a2) Prepare a kit for identifying *Basilella marneffei*;

[0034] (a3) Detect whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0035] (a4) Prepare a kit for detecting whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0036] (a5) Detect whether the sample to be tested contains *Basilella marneffei*;

[0037] (a6) Prepare a kit for detecting whether a sample contains *Bambusa marneffei*.

[0038] The present invention also protects a detection reagent or kit containing monoclonal antibody A5 of *Cladosporium marneffei* colp6, wherein the application of the detection reagent or kit is any one of the following (b1) to (b3):

[0039] (b1) Identification of *Basilella marneffei*;

[0040] (b2) To determine whether the pathogenic microorganism to be tested is *Basilella marneffei*;

[0041] (b3) Detect whether the sample to be tested contains *Bambusa marneffei*.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] This invention successfully cloned the light and heavy chain variable region gene sequences and amino acid sequences of the monoclonal antibody A5 for *Cladosporium marneffei* colp6. The monoclonal antibody A5 with the above-mentioned light and heavy chain variable regions can specifically recognize *Cladosporium marneffei*. The monoclonal antibody A5 obtained by this invention can bind to *Cladosporium marneffei* colp6, and has high antibody purity, strong specificity, and high detection sensitivity.

[0044] Instruction manual illustrations

[0045] Figure 1 Electrophoresis image of the Colp6 monoclonal antibody; in Figure 1 In the Maker lane, the bands from top to bottom represent the molecular weights as follows: 116KD, 66.2KD, 45KD, 35KD, 25KD, 18.5KD, and 14.5KD.

[0046] Figure 2 The graph shows the titer determination of monoclonal antibody A5.

[0047] Figure 3 Western blotting image of the Colp6 monoclonal antibody. Detailed Implementation

[0048] The following detailed description is provided in conjunction with specific embodiments, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, all raw materials used in the embodiments are commercially available.

[0049] Example 1: Preparation of monoclonal antibody against *Basilella marneffei* colp6

[0050] Step S1, Synthesis of the Colp6 gene

[0051] The amino acid sequence of the *Marneffeius* Colp6 gene was obtained from the GenBank sequence database. Codon optimization of the Colp6 gene base sequence was performed based on the codon bias of *E. coli*. The optimized gene sequence was then synthesized by Sangon Biotech (Shanghai) Co., Ltd. The Colp6 gene encodes 220 amino acid residues.

[0052] Colp6 amino acid sequence (SEQ ID NO.6);

[0053] MKFLPSLVVLGLSTQALASSYVDYVTKDQHGLTVYEMVINIINTTTSDFNTRIQSYQGGDLSSILEGCNQVTQIMKLGATILDQQTTKPLTNNEWLSLLSHMEKKGGLEDMLKMAINTLILKKSLILDSGLGSKLGLALYSQQMASIDLGAKFFEKTPPGKVEDFREHWFKNIMWVIGRGVDTFDKSTHHATIPTLPPRGAMATSNSPAIPTFTDAASAN

[0054] Colp6 nucleotide sequence (SEQ ID NO.7):

[0055] ATGAAGTTCTTACCCTCTCTCGTCGTCCTCGGTCTTTCTACCCAGGCTCTTGCGAGCTCTTACGTCGATTATGTTACTAAGGACCAGCATGGTCTTACTGTTTATGAGATGGTCATAAATATCATTAACACCACTACCTCAGACTTCAATACCCGGATTCAGAGTTACCAGGGTGGTGATCTCAGCAGCATTCTCGAAGGCTGTAATCAAGTTACCCAAATAATGAAGCTTGGGGCTACGATACTCGATCAGCAAACAACGAAACCCCTTACCAATAATGAGTGGCTTAGCCTCCTCTCACATATGGAGAAAAAGGGTGGTTTAGAAGATATGTTGAAAATGGCTATAAATACTCTTATCCTGAAGAAGTCACTTATTCTTGATTCTGGTTTGGGTTCTAAGCTTGGGTTAGCGCTTTACAGTCAGCAGATGGCTTCTATAGACCTCGGTGCTAAGTTCTTTGAGAAGACCCCCCCAGGAAAGGTCGAAGATTTCAGAGAACACTGGTTTAAGAACATCATGTGGGTCATCGGGCGAGGTGTTGATACCTTTGATAAATCTACCCATCACGCGACAATACCTACCTTACCCCCTAGAGGTGCTATGGCGACTAGTAATTCGCCTGCTATCCCTACTTTTACTGATGCTGCTAGTGCTAACTAG

[0056] Step S2: Expression and purification of the Colp6 gene

[0057] The Colp6 gene was ligated into the pQE 80L vector to obtain a recombinant vector. The plasmid of the recombinant vector was extracted and transformed into E. coli BL21(DE3). Expression was induced by IPTG. The bacterial cells were collected, high-pressure lysed, and the supernatant was collected after centrifugation. The protein was purified by Ni column affinity chromatography. The purified protein was further purified using a Superdex-200 column to obtain a high-purity Colp6 recombinant protein. The Colp6 recombinant protein was collected as an antigen for later use.

[0058] Step S3, mouse immunization

[0059] Purified Colp6 recombinant protein and Marneffei bacteria were used as immunogens and antigens. Three female BALB / c mice aged 6-8 weeks were immunized. Ten days after routine immunization, blood was collected from the tail vein and the antibody titers produced by the mice were detected by indirect ELISA. Three days before fusion, the recombinant antigen was injected for the last time via the tail vein at a dose of 60 μg / mouse for a booster immunization.

[0060] The immunization method for mice was as follows: 60 μg of the original emulsion was used per mouse, with a total volume of 350 μL of antigen + 1×PBS, mixed with an equal volume of Freund's adjuvant and emulsified 3500 times at 200 times / min. For the first immunization: 0.2 mL of the emulsion containing 60 μg of antigen was subcutaneously injected into the groin of each mouse using a 2 mL syringe, and the injection time and site were recorded. For the second immunization: two weeks later, 0.2 mL of the emulsion containing 60 μg of antigen was intraperitoneally injected into each mouse using a 2 mL syringe, and the injection time and site were recorded. For the third immunization: two weeks later, 0.2 mL of the emulsion containing 60 μg of antigen was subcutaneously injected into the groin of each mouse using a 2 mL syringe, and the injection time and site were recorded.

[0061] Step S4, Preparation of NS-1 myeloma cells

[0062] One week prior to fusion, NS-1 myeloma cells preserved in liquid nitrogen were revived and cultured in 25 cm⁻¹ medium. 2 In cell culture flasks, cells were passaged in DMEM medium containing 15% fetal bovine serum for one week, and the cell concentration was adjusted to 10⁻⁶. 6 Cells / mL; during fusion, select myeloma cells in the logarithmic growth phase, discard the original culture medium in the bottle, add an appropriate amount of serum-free DMEM culture medium, gently blow the cells off, transfer them to a 50mL centrifuge tube, centrifuge at 400g for 5 minutes, wash the cells 3 times, discard the supernatant, resuspend the cell pellet in serum-free culture medium, and count them for later use.

[0063] Step S5, Preparation of immune spleen cells

[0064] One BALB / c mouse with the best immune response was euthanized by bleeding from the eye socket and disinfected by soaking in 75% alcohol for 5 minutes. The disinfected mouse was fixed in a laminar flow hood, and the spleen was removed. Adhesive and connective tissue adhering to the cells were removed with scissors. The spleen was rinsed with serum-free culture medium and transferred to a 40μm cell filter. The cells were gently ground with a syringe core, and the filter was gently rinsed with serum-free culture medium. The spleen cell suspension was collected. The cells were centrifuged at 400g for 5 minutes, washed three times, and the supernatant was discarded. The cell pellet was resuspended in serum-free DMEM culture medium and counted for later use.

[0065] Step S6, Cell Fusion

[0066] Mix NS-1 myeloma cells and immune spleen cells at a 1:10 ratio in a 50 mL centrifuge tube, centrifuge at 400 g for 10 min, aspirate the supernatant, and gently tap the bottom of the centrifuge tube to loosen the cell pellet slightly. Slowly add 1 mL of 50% PEG solution pre-warmed to 37°C over 1 min, continuously and gently stirring the cells with a pipette for 1 min. Then add 10 mL of DMEM medium to the cell mixture, adding 1 mL dropwise over the first minute, 1 mL over the second minute, 3 mL over the third and fourth minutes, and the remaining 5 mL over the fifth minute. Incubate the cell mixture in a 37°C water bath for 15 min. Centrifuge at 400 g for 5 min, remove the supernatant, resuspend the cell pellet in 20 mL of 15% fetal bovine serum DMEM medium, and transfer to a 75 cm⁻¹ centrifuge tube. 2 Incubate the cells in a cell culture flask for 16–24 hours. Transfer the fused cell suspension to a 50 mL centrifuge tube and centrifuge at 400 g for 10 min. Remove the supernatant and resuspend the cell pellet in 2.5 mL of 15% fetal bovine serum DMEM medium. Add 22.5 mL of semi-solid medium, mix well, and pour into 3.5 cm diameter petri dishes, about 2 mL per dish. Incubate at 37 °C with 5% CO2. After ten days, visible white cell clones will appear on the surface of the culture medium in the petri dishes.

[0067] Step S7, Screening for positive hybridoma cells

[0068] Under aseptic conditions, cell clusters were aspirated from petri dishes and placed into 96-well plates for culture for 4 days. After 4 days, indirect ELISA was performed. Serum from normal mice before immunization was used as a negative control, and serum from mice after immunization was used as a positive control. A positive result was determined by an absorbance value 2.1 times greater than that of the negative control. Wells showing specific antibodies and exhibiting single-clone growth with good morphology were re-cloned. After at least 3 re-cloning, the hybridoma cells from the positive wells were transferred to 24-well plates. Once the hybridoma cells in the 24-well plates were growing well, the hybridoma cells were cryopreserved.

[0069] The indirect ELISA detection method is as follows: the antigen is coated onto the microplate at 100 ng / well, incubated overnight at 4°C, washed three times, 200 mL of blocking buffer is added, and the plate is incubated at 37°C for 2 h, washed three times, then hybridoma cell culture supernatant is added, and the plate is incubated at 37°C for 2 h, washed three times with washing buffer, then goat anti-mouse IgG-HRP diluted 1:10000 is added, and the plate is incubated at 37°C for 1 h, washed three times, and then developed at 37°C in the dark for 10 min. After sufficient development, 2 M H2SO4 is added to terminate the reaction, and the absorbance (OD value) is measured at a wavelength of 450 nm using a microplate reader.

[0070] Step S8, Preparation and purification of monoclonal antibodies

[0071] 0.5 mL of liquid paraffin was injected into the peritoneal cavity of 8-week-old BALB / c mice; hybridoma cells were then injected at a concentration of 1 × 10⁻⁶. 5 Inoculate at a concentration of cells / ml at a 25cm depth. 2 In a culture flask, culture until the cells reach optimal viability, then adjust the cell count to approximately 1×10⁶. 6 The ascites was inoculated into the peritoneal cavity of mice that had been injected with liquid paraffin. Ascites fluid was collected 7–10 days later. The ascites fluid was diluted more than 3 times with 20 mM PBS (pH 7.4), centrifuged, and the supernatant was purified by HiTrap protein G affinity chromatography. The purification method for the Protein G affinity chromatography column was as follows: a new column was first passed through 5 mL of ultrapure water, then equilibrated with 5 mL of 0.4 M PB buffer (pH 7.0). The antibody was then passed through the column slowly to ensure better binding of the antibody protein to the binding site. The column was then equilibrated again with 10 mL of 0.4 M PB buffer (pH 7.0). The antibody was then removed from the binding site with 5 mL of 0.1 M glycine-hydrochloric acid buffer (pH 2.7), and 1 M Tris-HCl (pH 8.0) was added to neutralize the glycine, maintaining a neutral pH suitable for antibody preservation. Two purified Colp6 monoclonal antibodies (A5 and A7) were obtained and subjected to protein electrophoresis with a loading volume of 1 μL. The results showed that both A5 and A7 antibodies had high purity, and the light and heavy chains of the antibodies could be observed after washing (see...). Figure 1 ).

[0072] Step S9, Sequencing of monoclonal antibodies

[0073] Hybridoma cells containing antibodies A5 and A7 were sent to Nanjing GenScript Biotech Co., Ltd. for antibody sequencing. The method was as follows: Total RNA was isolated from hybridoma cells according to the RNeasy Plus Micro Kit manual. Then, according to the SMARTScribe reverse transcriptase manual, the total RNA was reverse transcribed into cDNA using homotype-specific antisense primers or universal primers. Heavy and light chain antibody fragments were amplified according to the standard GenScript cDNA end amplification (RACE) procedure. The amplified antibody fragments were cloned into standard cloning vectors. Colony PCR was performed to screen clones with the correct-sized insert fragments.

[0074] The sequencing results of monoclonal antibody A5 are as follows:

[0075] The amino acid sequence of the light chain variable region of monoclonal antibody A5 is (SEQ ID NO.1): QIVLTQSPAILSASPGEKVTMTCRASSSVGYMHWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGAGTKLELKR

[0076] The amino acid sequence of the heavy chain variable region of monoclonal antibody A5 is (SEQ ID NO.2): EVKLVESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSGIISYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCARSGPWFAYWGQGTLVTVSA

[0077] The light chain variable region and the heavy chain variable region are linked by a short peptide, the amino acid sequence of which is (SEQ ID NO. 3):

[0078] RGGGGSGGGGSGGGG

[0079] Example 2: Titer determination of monoclonal antibody A5

[0080] Colp6 protein was coated with 100 ng / well. Monoclonal antibody A5 was diluted at 1:2000, 1:4000, 1:8000, 1:16000, 1:320000, 1:640000, 1:1280000, 1:2560000, 1:512000, 1:1024000, and 1:4096000 (original antibody concentration 1 mg / mL). The A450nm value was determined by indirect ELISA. The highest dilution of the monoclonal antibody that reacted with the target antigen was considered the titer. A positive result was defined as a ratio of the assay reading to the negative control value greater than 2.1. The specific procedure was as follows: coating with antigen, incubated overnight at 4°C. Blocking was performed with 3% BSA at 37°C for 2 hours; incubation was then performed with Colp6 monoclonal antibody as the primary antibody at 37°C for 1 hour; and with HRP-goat anti-mouse IgG as the secondary antibody at 37°C for 1 hour. Incubation was then performed with TMB chromogenic solution for 5 minutes; and finally stopped with 2mM sulfuric acid. The results showed that the titer of monoclonal antibody A5 was above 2 million (see...). Figure 2 )

[0081] Example 3: Specificity identification of monoclonal antibody A5

[0082] The specificity of the antibody was detected by Western blotting using Colp6 protein as the antigen. The specific experimental procedure was as follows: 1 μg of Colp6 protein was loaded onto a PVDF membrane via protein electrophoresis. The membrane was removed, washed with TBS, and blocked overnight at 4°C with 3% BSA for 5 min × 3. Purified monoclonal antibody was added at a ratio of 1:50000 (antibody concentration 0.02 μg / ml), and incubated for 1 h. The membrane was washed with TBST for 5 min × 3. HRP-labeled goat anti-mouse IgG antibody (secondary antibody) diluted 1:10000 was added, and incubated for 1 h. The membrane was washed with TBST for 5 min × 3. ECL chromogenic buffer was prepared according to a 1:1 ratio of A / B solutions and exposed on a chemiluminescence analyzer. Western blotting results showed that monoclonal antibody A5 could bind to Colp6 protein (see...). Figure 3 ).

[0083] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A monoclonal antibody A5 against *Basilella marneffei* colp6, characterized in that, The protein sequence of the monoclonal antibody A5 contains a light chain variable region and a heavy chain variable region. The light chain variable region has the amino acid sequence shown in SEQ ID NO.1, and the heavy chain variable region has the amino acid sequence shown in SEQ ID NO.

2. The light chain variable region and the heavy chain variable region are connected by a short peptide, the amino acid sequence of which is shown in SEQ ID NO.

3.

2. The monoclonal antibody A5 according to claim 1, characterized in that: The nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO.4, and the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.

5.

3. A detection reagent or kit containing the monoclonal antibody A5 as described in claim 1.

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