Human-mouse chimeric monoclonal antibody of anti-human Hu protein and application of human-mouse chimeric monoclonal antibody

Abstract

The invention provides an anti-human Hu protein human-mouse chimeric monoclonal antibody and application thereof, and belongs to the technical field of immunodetection. According to the anti-human Hu protein human-mouse chimeric monoclonal antibody provided by the invention, variable regions of a heavy chain and a light chain come from a mouse, the parent activity is reserved to the maximum extent, a constant region comes from a human source, and due to introduction of the human antibody constant region, the immunogenicity is greatly reduced, the occurrence probability of human anti-mouse antibody reaction is reduced, and the specificity of the antibody is improved. The human-mouse chimeric monoclonal antibody disclosed by the invention not only has antigen binding specificity, but also greatly reduces the heterology of the mouse monoclonal antibody. The chimeric antibody can recognize Hu protein, has biological activity, can be used for Hu antigen detection and other scientific researches, and has very good application value and very important scientific research guiding significance.

Description

Technical Field

[0001] The invention belongs to the technical field of immune detection, and in particular relates to human-mouse chimeric monoclonal antibodies against human Hu protein and applications thereof. Background Art

[0002] Paraneoplastic neurological syndrome (PNS) is a special autoimmune disease of the nervous system. Its mechanism is an immune-mediated tumor afar effect. Anti-neuronal antibodies play the most important role in the pathogenesis and can be used as diagnostic indicators and detected in the patient's serum or cerebrospinal fluid. PNS has a variety of autoantibodies, the most common of which are Hu antibodies, Yo antibodies, Ri antibodies, etc. These antibodies specifically bind to antigens in neuronal cells and can therefore be used as markers for PNS diseases.

[0003] Hu is also known as type 1 anti-neuronal nuclear antibody or ANNA-1. Its protein family members include HuA, HuB, HuC and HuD. HuA is widely expressed throughout the body, while HuB, HuC and HuD are specifically expressed in neurons and are located in the nucleus or cytoplasm. Anti-Hu autoantibodies were originally isolated from a patient with paraneoplastic encephalomyelitis. Currently, most of the detection of Hu autoantibodies is based on the detection of HuD autoantibodies, assisted by HuB / HuC autoantibodies. The traditional detection method is to use immunoblotting to detect whether the patient's serum contains autoantibodies that bind to the antigen coated on the test strip to determine whether the patient has the corresponding disease. Most test kits do not provide positive controls, or the positive controls used are mostly positive patient serum. Therefore, the kits cannot meet the needs of long-term positive control experiments, or the positive controls are not easy to obtain. Therefore, a human-mouse chimeric antibody against human Hu protein is produced as a positive control, which can not only meet the needs of positive control experiments, but also be provided stably for a long time, with good specificity and high titer. Summary of the invention

[0004] The present invention provides human-mouse chimeric monoclonal antibodies against human Hu protein and applications thereof. The human-mouse chimeric monoclonal antibody carrier is easy to store and easy to control the quality of the antibody production process. The human-mouse chimeric monoclonal antibody can recognize Hu protein and has biological activity.

[0005] The object of the present invention is to provide a human-mouse chimeric monoclonal antibody against human Hu protein, the structure of which includes a heavy chain and a light chain;

[0006] The light chain includes a light chain variable region and a light chain constant region, wherein the light chain variable region includes a complementary determining region as shown in amino acid sequences such as SEQ ID No.2 to SEQ ID No.4, and the light chain constant region is a kappa type human light chain constant region;

[0007] The heavy chain includes a heavy chain variable region and a heavy chain constant region, wherein the heavy chain variable region includes a complementary determining region as shown in the amino acid sequence of SEQ ID No. 7 to SEQ ID No. 9, and the heavy chain constant region is a human IgG1 heavy chain constant region.

[0008] In a specific embodiment of the present invention, the light chain variable region comprises the amino acid sequence shown in SEQ ID No.1.

[0009] In a specific embodiment of the present invention, the nucleotide sequence encoding the light chain variable region is shown as SEQ ID No.5.

[0010] In a specific embodiment of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.6.

[0011] In a specific embodiment of the present invention, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID No.10.

[0012] In a specific embodiment of the present invention, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 11;

[0013] The amino acid sequence of the heavy chain constant region is shown in SEQ ID No.12.

[0014] A further object of the present invention is to provide a recombinant vector for expressing the human-mouse chimeric monoclonal antibody.

[0015] A further object of the present invention is to provide a recombinant cell expressing the human-mouse chimeric monoclonal antibody.

[0016] A further object of the present invention is to provide the use of the human-mouse chimeric monoclonal antibody in the preparation of a reagent for specifically detecting human Hu autoantibodies.

[0017] A further object of the present invention is to provide the use of the human-mouse chimeric monoclonal antibody in the preparation of a reagent for the diagnosis and / or prognosis of paraneoplastic syndrome.

[0018] Beneficial effects: The present invention provides a human-mouse chimeric monoclonal antibody against human Hu protein, that is, a monoclonal antibody produced by replacing the constant region gene of a mouse monoclonal antibody with the constant region gene of a human antibody through gene recombination technology and encoding and expressing it in a suitable host cell, so that the variable regions of its heavy chain and light chain are from mice, retaining the parent activity to the maximum extent, and the constant region is from human, and the introduction of the human antibody constant region greatly reduces the immunogenicity, reduces the probability of human anti-mouse antibody reaction, and improves the specificity of the antibody. The human-mouse chimeric monoclonal antibody of the present invention has both antigen binding specificity and greatly reduces the heterogeneity of mouse monoclonal antibodies.

[0019] In one embodiment of the present invention, the coding gene of the human-mouse chimeric monoclonal antibody is constructed into an antibody expression vector, and the cells are transfected to express the recombinant human-mouse chimeric antibody. The chimeric antibody vector obtained by the present invention is easy to store and easy to control the quality of the antibody production process; it is also convenient to make a series of modifications to the antibody, more in-depth research, and wider application. It has been verified by the examples that the chimeric antibody of the present invention or the chimeric antibody produced by using recombinant cells can recognize Hu protein, has biological activity, can be used for other scientific research such as Hu antigen detection, and has very good application value and very important scientific research guidance significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The graphs are the results of HuD-6his and HuB-6his protein expression;

[0021] Figure 2 This is the titer result diagram of Hu antigen immunized mice;

[0022] Figure 3 This is a diagram showing the typing and identification results of the 10A8 mouse monoclonal antibody;

[0023] Figure 4 This is the result of protein expression of Hu-10A8 human-mouse chimeric monoclonal antibody;

[0024] Figure 5 This is a diagram showing the titer determination results of Hu-10A8 human-mouse chimeric monoclonal antibody;

[0025] Figure 6 This is a dot blot analysis to verify the function of the anti-Hu human-mouse chimeric monoclonal antibody;

[0026] Figure 7 This is a diagram showing the functional results of the anti-human Hu human-mouse chimeric monoclonal antibody verified by western blot. DETAILED DESCRIPTION

[0027] The present invention provides a human-mouse chimeric monoclonal antibody against human Hu protein, the structure of which includes a heavy chain and a light chain;

[0028] The light chain includes a light chain variable region and a light chain constant region, wherein the light chain variable region includes a complementary determining region as shown in amino acid sequences such as SEQ ID No.2 to SEQ ID No.4, and the light chain constant region is a kappa type human light chain constant region;

[0029] The heavy chain includes a heavy chain variable region and a heavy chain constant region, wherein the heavy chain variable region includes a complementarity determining region as shown in the amino acid sequence of SEQ ID No.7 to SEQ ID No.9, and the heavy chain constant region is a human IgG1 heavy chain constant region.

[0030] The human-mouse chimeric monoclonal antibody described in the present invention has light chain variable regions and heavy chain variable regions both from mice, which can retain the parent activity to the maximum extent; the light chain constant region and heavy chain constant region both from humans, and the introduction of humanized antibody constant regions can reduce immunogenicity, reduce the probability of human anti-mouse antibody reaction, and improve the specificity of the antibody.

[0031] The amino acid sequences of the complementary determining regions (CDR regions) of the variable regions of the light chains of the present invention may be: CDR1 (SEQ ID No. 2): RASQEISGYLS;

[0032] CDR2(SEQ ID No.3): STSTLDS;

[0033] CDR3 (SEQ ID No. 4): LQYVSSPLT.

[0034] In one embodiment of the present invention, the amino acid sequence of the light chain variable region is shown in SEQ ID No. 1:

[0035] MDMRVPAHVFGFLLFWFPGARCDIQMTQSPSSLSASLGERVSLTCRASQ EISGYLSWLQQKPDGTIKRLIYSTSTLDSGVPKRFSGSRSGSDYSLTISSLESED FADYYCLQYVSSPLTFGAGTKLELK, the bold font is the CDR region; the nucleotide sequence encoding the light chain variable region used in the embodiment can be shown as SEQ ID No.5.

[0036] The amino acid sequences of the complementarity determining regions (CDR regions) of the heavy chain variable regions of the present invention may be: CDR1 (SEQ ID No. 7): SYWMH;

[0037] CDR2 (SEQ ID No. 8): NINPSNGGTNYNEKFKS;

[0038] CDR3 (SEQ ID No. 9): DGNNDYWFFDV.

[0039] In one embodiment of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.6:

[0040] MGWSCIILFLVAAATGVHSQVQLQQPGTELVKPGASVKLSCKASGYIFTS YWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSNTAY MQVSSLTSEDSAVYYCARDGNNDYWFFDVWGAGTTVTVSS, the bold font is the CDR region; in the embodiment, the nucleotide sequence encoding the heavy chain variable region can be shown as SEQ ID No.10.

[0041] The human-mouse chimeric monoclonal antibody of the present invention also includes a constant region, wherein the light chain constant region is a kappa type. In one embodiment, the amino acid sequence of the human kappa type light chain constant region is shown as SEQ ID No.11; in the embodiment, the nucleotide sequence encoding the light chain constant region can be shown as SEQ ID No.13.

[0042] The heavy chain constant region of the present invention is a human IgG1 heavy chain constant region. In one embodiment, the amino acid sequence of the heavy chain constant region is shown as SEQ ID No.12; in the embodiment, the nucleotide sequence encoding the heavy chain constant region can be shown as SEQ ID No.14.

[0043] A further object of the present invention is to provide a recombinant vector for expressing the human-mouse chimeric monoclonal antibody.

[0044] The recombinant vectors described in the present invention and in the embodiments are based on pcDNA3.1 as the basic backbone vector, and the light chain variable region and the light chain constant region are respectively connected to the vector pcDNA3.1, named pcDNA3.1-Hu-10A8-VL, wherein the complete sequences of the light chain variable region and the light chain constant region are shown in SEQ ID No.15; the heavy chain variable region and the heavy chain constant region are connected to the vector pcDNA3.1, named pcDNA3.1-Hu-10A8-VH, wherein the complete sequences of the heavy chain variable region and the heavy chain constant region are shown in SEQ ID No.16.

[0045] A further object of the present invention is to provide a recombinant cell expressing the human-mouse chimeric monoclonal antibody.

[0046] The host cell of the recombinant cell of the present invention can be a mammalian cell, and in one embodiment, it is an Expi293F cell. The pcDNA3.1-Hu-10A8-VL plasmid and the pcDNA3.1-Hu-10A8-VH plasmid are co-transfected into Expi293F cells for transient expression to obtain cells transfected with the human-mouse chimeric monoclonal antibody plasmid against human Hu protein.

[0047] The present invention also provides a method for producing the human-mouse chimeric monoclonal antibody using the recombinant cells, which specifically comprises the steps of transfecting the cells transfected with the human-mouse chimeric monoclonal antibody plasmid against human Hu protein using OPM-293CD05 culture medium (manufacturer: OPM), collecting the supernatant after 3 days, and purifying them using protein A filler to obtain the human-mouse chimeric monoclonal antibody.

[0048] A further object of the present invention is to provide the use of the human-mouse chimeric monoclonal antibody in the preparation of a reagent for specifically detecting human Hu autoantibodies.

[0049] The human-mouse chimeric monoclonal antibody of the present invention can specifically recognize HuB and HuD antigens, and has substantially the same activity as the mouse monoclonal antibody. The monoclonal antibody of the present invention can be used to specifically detect Hu protein, has biological activity, is easy to store, and is easy to control the quality of the antibody production process; it can be used as a positive quality control in the detection of human Hu autoantibody reagents to verify the expression of Hu protein.

[0050] A further object of the present invention is to provide the use of the human-mouse chimeric monoclonal antibody in the preparation of a reagent for the diagnosis and / or prognosis of paraneoplastic syndrome.

[0051] The human-mouse chimeric monoclonal antibody of the present invention can be used as a substitute for positive serum to meet the diagnostic and / or prognostic reagents of the present invention, especially the requirements of positive controls in the kit. The kit for diagnosis and / or prognostic judgment of paraneoplastic syndrome of the present invention may contain Hu protein pre-coated on a test strip, working solution and washing solution, enzyme-labeled (horseradish peroxidase / alkaline phosphatase) anti-human IgG secondary antibody, color developing solution (TMB / BCIP-NBT), and a dot blot method is used to detect whether the patient's serum contains autoantibodies that bind to the antigen coated on the test strip, and the enzyme-labeled secondary antibody recognizes the antibody specifically bound to the test strip, and finally develops color through the enzyme-substrate to form a visible color spot to determine whether the patient contains the corresponding disease. The monoclonal antibody of the present invention can be used as a substitute for positive serum, and plays a positive quality control role in the contents of the kit during the detection process.

[0052] To further illustrate the present invention, the human-mouse chimeric monoclonal antibody against human Hu protein provided by the present invention and its application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] Expression and purification of Hu prokaryotic protein

[0055] The human HuB gene sequence was obtained from the GenBank sequence database, with the sequence number NM_004432.5, and the human HuD gene sequence was obtained, with the sequence number NM_021952.5. By comparing the homology of the human HuB protein and the HuD protein, it was found that the sequence homology between the 6th to 356th position of the HuB protein amino acid sequence and the 13th to 377th position of the HuD protein amino acid sequence reached 90.5%. Therefore, the full-length amino acid sequence of the HuD protein was selected, and a 6his tag sequence was added to the C-terminus of the gene. The gene sequence was synthesized by Jinweizhi Company between BamHⅠ and NotⅠ of the pET32a vector, and a plasmid was constructed and named HuD-6his. The constructed and correctly sequenced plasmid was transferred into the expression competent cells of Escherichia coli BL21 (DE3); a single clone was picked and inoculated into LB medium, and the bacteria were shaken at 37°C until the OD 600 value of 0.5 to 1, induced by IPTG, expressed at 16°C overnight; the bacteria were collected, ultrasonically broken, and the supernatant was collected after centrifugation. The HuD protein expressed by pET-30a was purified by Ni column affinity chromatography, and the purified protein was recorded as HuD-6his; the purified protein was dialyzed and concentrated to obtain a high concentration of HuD-6his protein as an antigen for later use.

[0056] The same method was used to add a 6his tag sequence to the C-terminus of the human HuB gene sequence (NM_004432.5). The gene sequence was synthesized by Jinweizhi Company between BamHⅠ and NotⅠ of the pET32a vector, and a plasmid was constructed and named HuB-6his. It was expressed and purified, and a high-concentration HuD-6his protein was obtained after dialysis and concentration as a detection protein.

[0057] Depend on Figure 1 It can be seen that HuD-6his and HuB-6his proteins were expressed and purified successfully.

[0058] Example 2

[0059] Obtaining hybridoma cells

[0060] Step 1: Immunize mice with Hu antigen

[0061] 1. Mix 40 μg of purified HuD-6his antigen with complete Freund's adjuvant in a 1:1 ratio;

[0062] 2. Take 3 female Balb / c mice aged 6 to 8 weeks, and inject 100 μL (40 μg antigen) of the mixed antigen into the left hind calf muscle; 14 days after the first immunization, perform the second immunization, 40 μg antigen mixed with incomplete Freund's adjuvant in a ratio of 1:1, and inject 100 μL (40 μg antigen) of the mixed antigen into the right hind calf muscle. 14 days after the second immunization, perform the third immunization, and the dosage, method and route of the injection of antigen are exactly the same as those of the second immunization;

[0063] Step 2: ELISA to detect antibody production in mice

[0064] Seven days after the third immunization, blood was collected from the tail of the mice, and serum was collected by centrifugation. ELISA was used to detect the antibody production of the mice. The specific steps are as follows:

[0065] ① Use purified HuB-6his and HuD-6his proteins to coat one ELISA plate respectively, 100 ng / well, overnight coating at 4°C, coating solution: 25 mL carbonate buffer, pH 9.6 (Na2CO3 0.03975 g, NaHCO3 0.07325 g, KH2PO4 0.00625 g);

[0066] ② Wash 3 times with PBST, 3 min each time, and pat dry each time;

[0067] ③ Block the wells with 2% BSA and incubate at 37°C for 1 h;

[0068] ④ Wash 3 times with PBST, 3 min each time, and pat dry each time;

[0069] ⑤ Dilute the serum with diluent in multiples, at 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350, add 100 μL to each well, incubate at 37°C for 1 hour, wash 3 times with PBST, 3 minutes each time, and pat dry each time;

[0070] ⑥ Sheep anti-mouse IgG-HRP 1:5000 dilution, incubate at 37℃ for 30min;

[0071] ⑦ Wash with PBST three times, pat dry, develop with TMB for 10 min, terminate with 2M H2SO4, and measure absorbance at 450 nm. Figure 2 The antibody titer of mouse No. 2 was above 109350 when the coating antigen was HuB protein, and the antibody titer was above 109350 when the coating antigen was HuD protein. Based on comprehensive analysis, mouse No. 2 was selected for subsequent experiments.

[0072] Step 3: Cell fusion

[0073] 1. Preparation of Myeloma Cells

[0074] SP2 / 0 cells were revived and subcultured in 15% fetal bovine serum DMEM medium for one week. Myeloma cells in the logarithmic growth phase were selected for fusion.

[0075] 2. Preparation of Splenocytes

[0076] Take No. 2 mouse with good immune effect, collect tail blood to separate serum, kill the mouse by cervical dislocation, and immerse in 75% alcohol for 5 minutes for disinfection; fix the mouse in the clean bench, remove the spleen, and use scissors to remove the fat tissue and connective tissue of the adhered cells; rinse the spleen with serum-free culture medium, place it on the cell filter, gently grind it with the inner core of the syringe, and gently rinse the filter with serum-free culture medium to collect the spleen cell suspension; centrifuge at 500g for 5 minutes, wash the cells 3 times, discard the supernatant, suspend the cell precipitate with serum-free DMEM culture medium, count and set aside.

[0077] 3. Cell Fusion

[0078] Prepare a 37℃ water bath in a clean bench, add SP2 / 0 cells and spleen cells in a 50mL centrifuge tube at a ratio of 1:10, and mix well. Centrifuge at 500g for 10 minutes, aspirate the supernatant, and tap the bottom of the centrifuge tube to loosen the cell pellet slightly; slowly add 1mL of 45% PEG1450 solution preheated to 37℃ within 90 seconds; and shake the centrifuge tube gently; the centrifuge tube is kept in a 37℃ water bath during the whole process.

[0079] Then gradually add DMEM medium to the cell mixture, drip 1 mL dropwise in the first minute, add 2 mL in the second minute, add 3 mL in the third minute, add 4 mL in the fourth minute, and add 5 mL in the fifth minute, shaking in a 37°C water bath. Then incubate at 37°C for 15 minutes, centrifuge at 500g for 5 minutes, and remove the supernatant.

[0080] Add 5 mL of DMEM medium containing HAT, gently suspend the precipitated cells, and finally add DMEM medium containing HAT to about 100 mL. Dispense into a 96-well cell culture plate with macrophages, 100uL / well, and then place the culture plate in a 37°C, 5% CO2 incubator for culture.

[0081] Step 3: ELISA detection of positive hybridoma cells

[0082] 1. Coat the ELISA plate with HuD-6his protein at 100 ng / well at 4°C overnight.

[0083] 2. Observe the growth of hybridoma cells. After seven days, when the cell culture supernatant turns yellow, take an appropriate amount of cell supernatant for ELISA antibody detection.

[0084] 3. Based on the ELISA results, select clones with high OD values ​​that are more than twice that of the negative control, plate them on a 96-well plate, and perform the first subclone screening.

[0085] 4. After seven to ten days, perform ELISA to detect antibodies again, select clones with high OD values, spread them on 96-well plates, and perform a second subclone screening to make each well have about 1 cell;

[0086] 5. After seven to ten days of culture, perform ELISA to detect antibodies, select clones with high OD values, spread 96-well plates, and perform the third subclone screening, so that each well has about 1 cell. Select several clones with high OD values ​​and perform ELISA detection. Finally, the positive clone cell with the highest value was screened out and named: 10A8 hybridoma cell, and the monoclonal antibody secreted by the cell was named 10A8 mouse monoclonal antibody.

[0087] Example 3

[0088] Identification of monoclonal antibody typing and sequence identification

[0089] Step 1. Use the 10A8 hybridoma cells screened in Example 2 to expand the culture and identify the antibody typing: use the re-coated purified HuD-6his protein as an antigen to detect the typing of the 10A8 mouse monoclonal antibody, the antigen coating concentration is 100 ng / well, and the coating is carried out at 4°C overnight; the next day, wash 3 times with 0.1MP BST, 3 minutes each time, and pat dry; add blocking protein (2% BSA solution), 200 μL / well, incubate at 37°C for 1 hour; wash 3 times with 0.1MP BST, 3 minutes each time, and pat dry; add 100 μL / well of monoclonal 10A8 cell supernatant, and use the tail blood of No. 2 mouse at a dilution of 1:1000 as a positive control As a negative control, pCDNA3.1 cell supernatant was used, 100 μL was added to each well, and the cells were incubated at 37°C for 1 h; the cells were washed three times with 0.1 M P BST for 3 min each time, and the cells were patted dry; HRP-labeled secondary antibodies (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / IgKappachain / Igglambdachain) were used, and the antibody was diluted 1:5000 (manufacturer: Jackson), and the cells were incubated at 37°C for 30 min; the cells were washed three times with 0.1 M P BST for 3 min each time, and the cells were patted dry; TMB was added for color development for 5 min, and the color development was terminated with 2 M H2SO4, and the absorbance was measured at 450 nm.

[0090] The experimental results are as follows Figure 3As shown, the heavy chain of 10A8 mouse monoclonal antibody is IgG1, and the light chain is Kappa chain. Then, 10A8 mouse monoclonal antibody was purified from the culture supernatant of positive hybridoma cells and used as positive control protein. The protein concentration was 0.6 mg / mL measured by BCA method. Subsequent experiments were continued with 10A8 hybridoma cells.

[0091] Step 2: 10A8 hybridoma cells were cultured and lysed with Trizol, and total RNA of hybridoma cell lysate was extracted (Quick-RNA MicroPrep Kit), RNA was separated by agarose gel electrophoresis, and then 5'RACE was performed to obtain cDNA (Clontech SMARTerRACE 5' / 3'kit), which was used as a template for PCR amplification ( MaxDNAPolymerase) was used to extract the antibody variable region cDNA, which was then sequenced by a sequencing company to obtain the amino acid sequence of the light chain variable region and the heavy chain variable region of the 10A8 mouse monoclonal antibody. SEQ ID No.1 is the amino acid sequence of the light chain variable region of the 10A8 mouse monoclonal antibody, and SEQ ID No.6 is the amino acid sequence of the heavy chain variable region of the 10A8 mouse monoclonal antibody. The antigen-determining complementary region (CDR) region of the amino acid sequence of the 10A8 mouse monoclonal antibody was marked using the Kabat method. SEQ ID No.5 is the nucleotide sequence of the light chain variable region of the 10A8 mouse monoclonal antibody, and SEQ ID No.10 is the nucleotide sequence of the heavy chain variable region of the 10A8 mouse monoclonal antibody.

[0092] Example 4

[0093] Preparation of anti-Hu human-mouse chimeric monoclonal antibodies

[0094] Step 1: Construction of recombinant vector

[0095] The nucleotide sequence of the light chain variable region of the anti-human Hu protein 10A8 mouse monoclonal antibody (SEQ ID No. 5) was amplified, and the nucleotide sequence of the human Kappa chain constant region (SEQ ID No. 13) was amplified. The two fragments were connected to the vector pcDNA3.1 using homologous recombination (manufacturer: Quanshijin Product No.: CU201-02), marked as pcDNA3.1-Hu-10A8-VL, and the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmid was extracted and used for cell transfection to prepare antibodies.

[0096] The primers for amplifying the sequence shown in SEQ ID No.5 are as follows:

[0097] Upstream primer F (SEQ ID No. 17): ctatagggagacccaagctggctagcATGGACATGAGGGTTCCTGC;

[0098] Downstream primer R (SEQ ID No. 18): agacagatggtgcagccacagttctTTTCAGCTCCAGCTTGGTCC;

[0099] The primers for amplifying the sequence shown in SEQ ID No.13 are as follows:

[0100] Upstream primer F (SEQ ID No. 19): tgctgggaccaagctggagctgaaaAGAACTGTGGCTGCACCATC;

[0101] Downstream primer R (SEQ ID No. 20): aacgggccctctagactcgagcggccgcCTAACACTCTCCCCTGTTGA;

[0102] The amplification procedures for SEQ ID No.5 and SEQ ID No.13 were: 95°C pre-denaturation for 5 min ； Denaturation at 95°C for 30 s, annealing at 62°C for 20 s, and extension at 72°C for 20 s, for 30 cycles; and extension at 72°C for another 3 min.

[0103] The nucleotide sequence of the heavy chain variable region of the anti-human Hu protein 10A8 mouse monoclonal antibody (SEQ ID No. 10) was amplified, and the human IgG1 heavy chain constant region sequence (SEQ ID No. 14) was amplified. The two fragments were connected to the vector pcDNA3.1 using homologous recombination (manufacturer: Quanshijin Product No.: CU201-02), marked as pcDNA3.1-Hu-10A8-VH, and the connected recombinant plasmid was sent to Sangon Biotechnology for sequencing. The sequenced recombinant plasmid was extracted and used for cell transfection to prepare antibodies.

[0104] The primers used to amplify the sequence shown in SEQ ID No.10 are as follows:

[0105] Upstream primer F (SEQ ID No. 21): ctatagggagacccaagctggctagcATGGGATGGAGCTGTATCAT;

[0106] Downstream primer R (SEQ ID No. 22): agaccgatgggcccttggtggaggcTGAGGAGACGGTGACCGT;

[0107] The procedure used to amplify the sequence shown in SEQ ID No.10 was: pre-denaturation at 95°C for 5 min ； Denaturation at 95°C for 30 s, annealing at 58°C for 20 s, extension at 72°C for 20 s, 30 cycles; and extension at 72°C for another 3 min.

[0108] The primers used to amplify the sequence shown in SEQ ID No.14 are as follows:

[0109] Upstream primer F (SEQ ID No. 23): agggactacggtcaccgtctcctcaGCCTCCACCAAGGGCCCATC;

[0110] Downstream primer R (SEQ ID No. 24): aacgggccctctagactcgagcggccgcTCATTTACCCGGAGACAGGGA.

[0111] The procedure used to amplify the sequence shown in SEQ ID No.14 was: pre-denaturation at 95°C for 5 min ； Denaturation at 95°C for 30 s, annealing at 64°C for 20 s, extension at 72°C for 30 s, 30 cycles; and extension at 72°C for another 3 min.

[0112] Step 2: Protein expression and purification

[0113] When the cell density reached 4.5 million / mL and the total cell volume was 100 mL, 175 μg of plasmid was co-transfected according to the ratio of heavy chain plasmid: light chain plasmid of 1:1, and the amount of PEI was three times the amount of plasmid, 525 μg. 87.5 μg of the reconstructed pcDNA3.1-Hu-10A8-VL plasmid and pcDNA3.1-Hu-10A8-VH plasmid were co-transfected into Expi293F cells for transient expression (transfection reagent PEI, thermo), and cells transfected with the human-mouse chimeric monoclonal antibody 10A8 plasmid against human Hu protein were obtained. The supernatant was collected 3 days after transfection, purified using protein A filler, and labeled as Hu-10A8. The Hu-10A8 protein concentration was determined by BCA method to be 0.4 mg / mL. Figure 4 As shown, Hu-10A8 human-mouse chimeric monoclonal antibody was successfully expressed and purified.

[0114] Example 5

[0115] Application of anti-human Hu human-mouse chimeric monoclonal antibodies

[0116] 1. ELISA verifies the function of anti-Hu human-mouse chimeric monoclonal antibodies

[0117] Use purified HuB-6his protein and HuD-6his protein to coat an ELISA plate, 100 ng / well, overnight at 4°C (the coating solution is 25 mL carbonate buffer, pH 9.6); wash with PBST solution 3 times the next day, 3 min each time, and pat dry each time; add 2% BSA to cover the wells, and incubate at 37°C for 1 hour; after incubation, wash with PBST 3 times, 3 min each time, and pat the plate to remove excess water each time; incubate the Hu-10A8 human-mouse chimeric antibody prepared in Example 4 and the 10A8 mouse monoclonal antibody prepared in Example 1 as positive For control, normal control mouse serum was used as negative control, and the dilution ratios were 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187 of the original solution, respectively. 100 μL of the above dilutions were added to each well, incubated at 37°C for 1 h, washed 3 times with PBST, 3 min each time, and patted dry each time; goat anti-human / goat anti-mouse IgG-HRP secondary antibody was diluted 1:5000, added to the plate, and incubated at 37°C for 30 min; after incubation, washed 3 times with PBST, patted dry, TMB was added for color development for 3 min, 2M H2SO4 was used for termination, and the absorbance was measured at 450 nm.

[0118] The experimental results are as follows Figure 5 As shown, the modified Hu-10A8 human-mouse chimeric monoclonal antibody can specifically recognize HuB and HuD antigens, and the activity of the modified Hu-10A8 human-mouse chimeric antibody is basically consistent with that of the 10A8 mouse monoclonal antibody before modification.

[0119] 2. Dot blot method to verify the function of anti-human Hu human-mouse chimeric monoclonal antibody

[0120] In order to verify whether the prepared human-mouse chimeric antibody has the ability to target and detect human HuB antigen and HuD antigen, the Hu-10A8 monoclonal antibody prepared and purified in Example 3 was used for dot blot test verification. The test steps are as follows:

[0121] Protein distribution Figure 6As shown, the quality control point (Ctrl) is an anti-human Fab antibody (manufacturer: Sigma) with a concentration of 100 ng / μL; HuB is HuB-6his protein, and HuD is HuD-6his protein, which are diluted to a concentration of 50 ng / μL. 0.8 μL of sample is taken using a micropipette, and is bound to a right-angled trapezoidal NC membrane in the form of a circular imprint spot, dried at 37° C. for 30 min, and placed in a 12-well plate, and incubated with the purified Hu-10A8 human-mouse chimeric monoclonal antibody (1:200 dilution) at a concentration of 0.4 mg / mL in Example 4, Hu antibody-positive (i.e., HuB antibody and HuD antibody are both positive) patient serum (1:10 dilution), Hu antibody-negative patient serum (1:10 dilution), anti-HuB+HuC+HuD commercial Antibody (1:1000 dilution, manufacturer: ABCAM), 10A8 mouse monoclonal antibody purified in Example 3 (1:200 dilution), 300ul / well, incubated on a shaker at room temperature for 30min, washed with 1% NaCl, 4min / time, washed twice in total; AP-labeled goat anti-human / mouse IgG secondary antibody diluted 2000 times (manufacturer: Jackson), added to the corresponding wells, incubated on a shaker at room temperature for 30min, washed with 1% NaCl, 4min / time, washed twice in total; after removing the washing solution in the well, add 50-fold diluted BCIP / NBT substrate, 300μL / well, stand at room temperature in the dark for 4min, discard the substrate dilution in the well, rinse the test strip with distilled water 1-2 times, take it out to dry or dry it at 37℃-45℃, observe the color development, and the results are as follows Figure 6 shown.

[0122] According to the results, it can be seen that the modified Hu-10A8 human-mouse chimeric antibody and Hu-positive patient serum can effectively recognize HuB and HuD antigens, as well as the quality control point (Ctrl) of anti-human Fab antibody, and form visible spots under the conditions of incubation with the same antigen and the same AP-labeled goat anti-human IgG secondary antibody, while the 10A8 mouse monoclonal antibody and the HuB+HuC+HuD commercial antibody are both mouse monoclonal antibodies, which need to be incubated with AP-labeled goat anti-mouse IgG secondary antibody to form visible spots, and do not recognize anti-human Fab antibodies, and cannot meet the requirements of quality control points. Due to the different species of incubated secondary antibodies and the recognition of quality control points, the 10A8 mouse monoclonal antibody and the HuB+HuC+HuD commercial antibody cannot meet the requirements as a substitute for patient serum, while the modified Hu-10A8 human-mouse chimeric antibody can be used as a substitute for positive serum to meet the requirements of the positive control in the diagnostic kit of the present invention.

[0123] 3. Western-Blot Identification of the Function of Anti-Hu Human-Mouse Chimeric Monoclonal Antibodies

[0124] The whole bacterial lysis liquid of the prokaryotic HuB and HuD constructed in Example 1 before and after induction was taken for use; then 1 dish of 293T cells overexpressing HuB, 1 dish of 293T cells overexpressing HuD and 1 dish of empty pCDNA3.1 cells were collected, lysed with RIPA lysis buffer, centrifuged to obtain the supernatant, and marked as HuB-pCDNA3.1, HuD-pCDNA3.1 and pCDNA3.1 for use; the above proteins were respectively subjected to gel electrophoresis, and after the electrophoresis, the membrane transfer conditions were 300 mA and 90 min for wet transfer; Block with 5% skim milk powder at room temperature for 1 hour; use 1:2000 diluted Hu-10A8 human-mouse chimeric antibody (concentration of 0.4 mg / mL) and commercial HuA / B / C / D (manufacturer: Wuhan Sanying, catalog number: 13032-1-AP) antibodies to incubate on the transferred PVDF membrane, and incubate overnight at 4°C; the next day, wash with TBST 3 times, 5 minutes each time; add HRP-labeled goat anti-human / goat anti-rabbit secondary antibody (manufacturer: Jackson), incubate at room temperature for 1 hour; wash with TBST 3 times, 5 minutes each time; add ECL chemiluminescent solution for color development and photography.

[0125] The results are as follows Figure 7 As shown, the modified Hu-10A8 human-mouse chimeric antibody can recognize HuB protein and HuD protein in overexpressed cells, and can also recognize HuB and HuD proteins after prokaryotic induced expression. Compared with commercial antibodies, it has obvious humanized species advantages and can be used as a serum substitute to meet the needs of long-term detection reagents.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A human-mouse chimeric monoclonal antibody against human Hu protein, characterized in that: The structure includes heavy and light chains; The light chain includes a light chain variable region and a light chain constant region, the light chain variable region includes a complementary determining region as shown in amino acid sequences such as SEQ ID No. 2 to SEQ ID No. 4, and the light chain constant region is a kappa type human light chain constant region; The heavy chain includes a heavy chain variable region and a heavy chain constant region. The heavy chain variable region includes a complementary determining region as shown in amino acid sequences such as SEQ ID No. 7 to SEQ ID No. 9, and the heavy chain constant region is a human IgG1 heavy chain constant region.

2. The human-mouse chimeric monoclonal antibody according to claim 1, characterized in that: The light chain variable region includes the amino acid sequence shown in SEQ ID No.

1.

3. The human-mouse chimeric monoclonal antibody according to claim 2, characterized in that: The nucleotide sequence encoding the light chain variable region is shown in SEQ ID No.

5.

4. The human-mouse chimeric monoclonal antibody according to claim 1, characterized in that: The heavy chain variable region includes the amino acid sequence shown in SEQ ID No.

6.

5. The human-mouse chimeric monoclonal antibody according to claim 4, characterized in that: The nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID No.

10.

6. The human-mouse chimeric monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the light chain constant region is shown in SEQ ID No.11; The amino acid sequence of the heavy chain constant region is shown in SEQ ID No.

12.

7. A recombinant vector expressing the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6.

8. A recombinant cell expressing the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6.

9. Use of the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6 in the preparation of a reagent for specifically detecting human Hu autoantibodies.

10. Use of the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6 in the preparation of a reagent for diagnosis and / or prognosis of paraneoplastic syndrome.

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