Human-mouse chimeric monoclonal antibodies against human hu proteins and uses thereof

Abstract

The application provides a human-mouse chimeric monoclonal antibody against human Hu protein and an application thereof, and belongs to the technical field of immune detection.The application provides a human-mouse chimeric monoclonal antibody against human Hu protein, the variable regions of the heavy chain and the light chain are from mice, the parent activity is maximally reserved, and the constant regions are from human sources;the introduction of the constant regions of human antibodies greatly reduces immunogenicity, reduces the probability of human anti-mouse antibody reaction, and improves the specificity of the antibody.The human-mouse chimeric monoclonal antibody has antigen binding specificity and greatly reduces the heterogeneity of mouse monoclonal antibodies.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 application belongs to the field of immunodetection technology, and particularly relates to a human-mouse chimeric monoclonal antibody against human Hu protein and application thereof. BACKGROUND

[0002] Paraneoplastic neurological syndrome (PNS) is a special autoimmune disease of the nervous system, and its mechanism is an immune-mediated tumor distant effect. Anti-neuron antibodies are the most important role in the pathogenesis process and can be used as a diagnostic indicator and are detected in the serum or cerebrospinal fluid of patients. PNS has various autoantibodies, and common ones are Hu antibody, Yo antibody, Ri antibody and the like. These antibodies are specifically combined with antigens in neuron cells, and thus can be used as markers of PNS diseases.

[0003] Hu is also called type 1 anti-neuron nuclear antibody or ANNA-1, and its protein family members include HuA, HuB, HuC and HuD. HuA is widely expressed in the whole body, while HuB, HuC and HuD are specifically expressed in neurons and are located in the nucleus or cytoplasm. Anti-Hu autoantibodies were initially isolated from a paraneoplastic myelitis patient. At present, the detection of Hu autoantibodies is mainly based on the detection of HuD autoantibodies, supplemented by HuB / HuC autoantibodies. The traditional detection method is to use an immunoblot method to detect whether the patient's serum contains autoantibodies combined with the antigens coated on the test strip, so as to determine whether the patient contains the corresponding disease. Most detection kits do not provide positive control products, or the positive control products used are mostly positive patient serum. Therefore, the kit cannot meet the long-term positive control experiment requirement, or the positive control product is not easy to obtain. Therefore, a human-mouse chimeric antibody against human Hu protein is produced as a positive control product, which can meet the positive control experiment requirement, is stably provided for a long time, and has good specificity and high titer. SUMMARY

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

[0005] The application aims to provide a human-mouse chimeric monoclonal antibody against human Hu protein, and the structure comprises a heavy chain and a light chain.

[0006] The light chain comprises a light chain variable region and a light chain constant region, wherein the light chain variable region comprises a complementarity determining region with an amino acid sequence as shown in SEQ ID No. 2-SEQ ID No. 4, and the light chain constant region is a kappa type human light chain constant region.

[0007] The heavy chain comprises a heavy chain variable region and a heavy chain constant region, wherein the heavy chain variable region comprises a complementarity determining region with an amino acid sequence as shown in SEQ ID No. 7-SEQ ID No. 9, and the heavy chain constant region is a human IgG1 heavy chain constant region.

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

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

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

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

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

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

[0014] It is a further object of the present application to provide a recombinant vector for expressing the above-mentioned human-mouse chimeric monoclonal antibody.

[0015] It is a further object of the present application to provide a recombinant cell for expressing the above-mentioned human-mouse chimeric monoclonal antibody.

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

[0017] It is a further object of the present application to provide the use of the above-mentioned human-mouse chimeric monoclonal antibody in the preparation of a reagent for the diagnosis and / or prognosis of paraneoplastic syndromes.

[0018] Beneficial effects: the present application provides a human-mouse chimeric monoclonal antibody against human Hu protein, i.e. a monoclonal antibody whose constant region genes of mouse-derived monoclonal antibody are replaced by constant region genes of human antibody through gene recombination technology, and the monoclonal antibody is produced by expression in a suitable host cell, so that the variable regions of the heavy chain and the light chain are from mice, the parent activity is maximally retained, and the constant region is from human source, the introduction of the constant region of human antibody 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 described in the present application has antigen binding specificity and greatly reduces the heterogeneity of mouse monoclonal antibody.

[0019] In one embodiment of the present application, the coding gene of the human-mouse chimeric monoclonal antibody is constructed into an antibody expression vector, and a cell is transfected to express a recombinant human-mouse chimeric antibody. The chimeric antibody vector obtained by the present application 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 more extensive application. It is verified by the embodiment that the chimeric antibody described in the present application or the chimeric antibody produced by the recombinant cell can recognize Hu protein, has biological activity, and can be used for Hu antigen detection and other scientific research, and has very good application value and very important scientific research guiding significance. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a HuD-6his and HuB-6his protein expression result graph;

[0021] Figure 2 is a Hu antigen immunized mouse titer result graph;

[0022] Figure 3 is a typing identification result graph of 10A8 mouse-derived monoclonal antibody;

[0023] Figure 4 is a Hu-10A8 human-mouse chimeric monoclonal antibody protein expression result graph;

[0024] Figure 5 is a Hu-10A8 human-mouse chimeric monoclonal antibody titer determination result graph;

[0025] Figure 6 is a dot blotting method verification result graph of the function of the anti-human Hu human-mouse chimeric monoclonal antibody;

[0026] Figure 7 is a western blot verification result graph of the function of the anti-human Hu human-mouse chimeric monoclonal antibody. DETAILED DESCRIPTION

[0027] The present application provides a human-mouse chimeric monoclonal antibody against human Hu protein, which has a structure comprising a heavy chain and a light chain;

[0028] The light chain comprises a light chain variable region and a light chain constant region, wherein the light chain variable region comprises a complementarity determining region with an amino acid sequence as shown in SEQ ID No. 2-4, and the light chain constant region is a kappa type human light chain constant region;

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

[0030] The human-mouse chimeric monoclonal antibody of the present application is derived from mice in the light chain variable region and the heavy chain variable region, which can maximize the retention of the parent activity; the light chain constant region and the heavy chain constant region are derived from humans, and the introduction of the human-derived antibody constant region can reduce the immunogenicity, reduce the probability of human anti-mouse antibody reaction, and improve the specificity of the antibody.

[0031] The amino acid sequence of the complementarity determining region (CDR region) of the variable region of the light chain of the present application can be: CDR1 (SEQ ID No. 2): RASQEISGYLS;

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

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

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

[0035] MDMRVPAHVFGFLLFWFPGARCDIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIKRLIYSTSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYVSSPLTFGAGTKLELK, the bolded part is the CDR region; the nucleotide sequence encoding the light chain variable region used in the embodiment can be shown in SEQ ID No. 5.

[0036] The amino acid sequence of the complementarity determining region (CDR region) of the heavy chain variable region of the present application can 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 application, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 6:

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

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

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

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

[0044] The recombinant vector of the present application is based on pcDNA3.1 as the backbone vector, and the light chain variable region and the light chain constant region are connected to the vector pcDNA3.1, named pcDNA3.1-Hu-10A8-VL, wherein the complete sequence of the light chain variable region and the light chain constant region is shown as 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 sequence of the heavy chain variable region and the heavy chain constant region is shown as SEQ ID No. 16.

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

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

[0047] The present application also provides a method for producing the human-mouse chimeric monoclonal antibody using the recombinant cell, which specifically comprises collecting the supernatant after the cell transfected with the human-mouse chimeric monoclonal antibody plasmid against human Hu protein is cultured for 3 days using the OPM-293CD05 medium (manufacturer: OPM), and purifying the supernatant using the protein A filler, so as to obtain the human-mouse chimeric monoclonal antibody.

[0048] The present application further provides an application of the human-mouse chimeric monoclonal antibody in preparing a reagent for specifically detecting the human Hu autoantibody.

[0049] The human-mouse chimeric monoclonal antibody of the present application can specifically recognize the HuB and HuD antigens, and has substantially the same activity as the mouse monoclonal antibody. The monoclonal antibody of the present application can be used to specifically detect the Hu protein, has the biological activity, is easy to preserve, and is easy to control the quality in the process of antibody production. The monoclonal antibody of the present application can be used as the positive control in the reagent for detecting the human Hu autoantibody, so as to verify the expression of the Hu protein.

[0050] The present application further provides an application of the human-mouse chimeric monoclonal antibody in preparing a reagent for diagnosing and / or judging the prognosis of the paraneoplastic syndrome.

[0051] The human-mouse chimeric monoclonal antibody of the present application can be used as the substitute of the positive serum, and meets the requirement of the positive control in the reagent, especially the reagent kit. The reagent kit for diagnosing and / or judging the prognosis of the paraneoplastic syndrome of the present application can contain the Hu protein pre-coated on the test strip, the working solution and the washing solution, the enzyme-labeled (horseradish peroxidase / alkaline phosphatase) anti-human IgG secondary antibody, and the color developing solution (TMB / BCIP-NBT). The spot blotting method is used to detect whether the patient serum contains the autoantibody combined with the antigen coated on the test strip, and the enzyme-labeled secondary antibody is used to recognize the antibody specifically combined with the test strip, and finally the enzyme-substrate is used for color development to form the color spot visible to the naked eye, so as to judge whether the patient contains the corresponding disease. The monoclonal antibody of the present application can be used as the substitute of the positive serum, and plays the role of the positive control of the content of the reagent kit in the detection process.

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

[0053] Example 1

[0054] Expression and purification of Hu prokaryotic protein

[0055] The human HuB gene sequence and the human HuD gene sequence were found in the GenBank sequence database, with the sequence number of NM_004432.5 and NM_021952.5. By comparing the homology of the human HuB protein and the human HuD protein, it was found that the sequence homology of the 6th-356th amino acid sequence of the HuB protein and the 13th-377th amino acid sequence of the HuD protein 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 terminal of the gene. The gene sequence was synthesized by the Jinweizhi Company between the BamH I and Not I of the pET32a vector, a plasmid was constructed and named as HuD-6his, and the constructed and sequenced plasmid was transferred into the E. coli BL21 (DE3) expression competent cells. A single colony was picked and inoculated in the LB medium, and the bacteria were shaken at 37°C until the OD value was 0.5-1. Then, IPTG was added for induction, and the expression was performed at 16°C overnight. The bacterial bodies were collected, and the supernatant was collected after ultrasonic disruption and centrifugation. The HuD protein expressed by the pET-30a was purified by Ni column affinity chromatography, and the purified protein was recorded as HuD-6his. After dialysis and concentration, the high-concentration HuD-6his protein was obtained as an antigen for standby use. 600

[0056] Using the same method, the 6his tag sequence was added to the C terminal of the human HuB gene sequence (NM_004432.5), and the gene sequence was synthesized by the Jinweizhi Company between the BamH I and Not I of the pET32a vector. A plasmid was constructed and named as HuB-6his, and the expression and purification were performed. After dialysis and concentration, the high-concentration HuD-6his protein was obtained as a detection protein for standby use.

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

[0058] Example 2

[0059] Obtaining of hybridoma cells

[0060] Step one, immunizing mice with Hu antigen

[0061] 1. 40 μg of the purified HuD-6his antigen was mixed with the complete Freund's adjuvant at 1:1;

[0062] ​​2. Take 3 6-8 weeks old female Balb / c mice, left hind leg muscle injection of 100 μL (40 μg antigen) mixed antigen; 14 days after the first immunization, the second immunization was carried out, 40 μg antigen mixed with incomplete Freund's adjuvant 1:1, right hind leg muscle injection of 100 μL (40 μg antigen) mixed antigen, 14 days after the second immunization, the third immunization was carried out, and the dose, method and route of injection were the same as the second immunization;

[0063] Step two, ELISA detection of mouse antibody production

[0064] Seven days after the third immunization, the mouse tail blood was collected, centrifuged to collect the serum, and the mouse antibody production was detected by ELISA, and the specific steps were as follows:

[0065] ①Use purified HuB-6his and HuD-6his protein to coat an ELISA plate respectively, 100 ng / well, 4℃ overnight coating, coating solution: 25 mL carbonate buffer, pH 9.6 (Na2CO30.03975 g, NaHCO30.07325 g, KH2PO40.00625 g);

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

[0067] ③2% BSA full well blocking, 37℃ incubation for 1h;

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

[0069] ⑤Dilute the serum with diluent by dilution ratio, dilute by 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350, add 100 μL to each well, 37℃ incubation for 1h, wash with PBST 3 times, 3 min each time, and pat dry each time;

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

[0071] ⑦PBST wash 3 times, pat dry, TMB color development for 10 min, 2M H2SO4 termination, 450nm measurement of absorbance value. Results are shown in Figure 2 , the antibody titer of No. 2 mouse is more than 109350 when the coating antigen is HuB protein, and the antibody titer is more than 109350 when the coating antigen is HuD protein, and the No. 2 mouse is selected for subsequent experiments.

[0072] Step three, cell fusion

[0073] 1. Preparation of myeloma cells

[0074] Recovery SP2 / 0 cells, with 15% fetal bovine serum DMEM culture medium subculture for a week. When the fusion, select the logarithmic growth of myeloma cells.

[0075] 2. Preparation of spleen cells

[0076] Take the immune effect of 2 mice, tail blood separation serum, and cervical dislocation lethal, 75% alcohol soak 5 min disinfection; in the clean bench, fixed mouse, remove the spleen, with scissors to remove the fat tissue and connective tissue of the cells; the spleen was washed with serum-free medium, placed on the cell filter, with a syringe core gently ground, and gently washed with serum-free medium filter, collect the spleen cell suspension; 500g centrifugation for 5 min, wash the cells 3 times, discard the supernatant, the cell pellet was suspended with serum-free DMEM medium, count for standby.

[0077] 3. Cell fusion

[0078] In the clean bench ready 37℃ water bath, SP2 / 0 cells and spleen cells were added to 50 mL centrifuge tube according to the proportion of 1:10, mixed evenly. 500g centrifugation for 10 min, suction supernatant, light off the bottom of the centrifuge tube, the cell pellet slightly loose; in 90 s slowly drop into the preheated to 37℃ 45% PEG1450 solution 1 mL; and constantly gently shake the centrifuge tube; the whole process of centrifuge tube in 37℃ water bath.

[0079] Then gradually add DMEM medium to the cell mixture, the first minute drop by drop into 1 mL, 2 min add 2 mL, 3 min add 3 mL, 4 min add 4 mL, 5 min add 5 mL, 37℃ water bath side add side shake. Then 37℃ incubation for 15 min, 500g centrifugation for 5 min, remove the supernatant.

[0080] Add 5 mL of DMEM medium containing HAT, gently suspended in the cell pellet, finally add HAT containing DMEM medium to about 100 mL. Dispensed in 96 well cell culture plate with macrophages, 100 uL / well, then the culture plate was placed in 37℃, 5% CO2 incubator.

[0081] Step three, ELISA detection of positive hybridoma cells

[0082] 1. ELISA plate coated with HuD-6his protein, 100 ng / well, 4℃ overnight coating.

[0083] 2. Observation of hybridoma cell growth, after seven days when the cell culture supernatant yellow, take the appropriate amount of cell supernatant for ELISA detection of antibody.

[0084] 3. According to the ELISA results, select the clones with high OD values, which are more than twice higher than the negative control, and plate them in 96-well plates for the first subcloning screening.

[0085] 4. After seven to ten days, perform ELISA detection of the antibodies again, select the clones with high OD values, and plate them in 96-well plates for the second subcloning screening, so that there are about 1 cell per well;

[0086] 5. After seven to ten days of culture, perform ELISA detection of the antibodies again, select the clones with high OD values, and plate them in 96-well plates for the third subcloning screening, so that there are about 1 cell per well. Select several clones with high OD values and perform ELISA detection. Finally, select the positive clone cell with the highest value, which is named 10A8 hybridoma cell. The monoclonal antibody secreted by the cell is named 10A8 murine monoclonal antibody.

[0087] Example 3

[0088] Identification of the typing of the monoclonal antibody and identification of the sequence

[0089] Step one, use the 10A8 hybridoma cell screened in Example 2 to perform large-scale culture and identify the typing of the antibody: use the purified HuD-6his protein coated again as the antigen to detect the typing of the 10A8 murine monoclonal antibody, the antigen coating concentration is 100 ng / well, and the coating is performed at 4°C overnight; the next day, wash 3 times with 0.1M PBST, each time for 3 min, and pat dry; add blocking protein (2% BSA solution), 200 μL / well, and incubate at 37°C for 1 h; wash 3 times with 0.1M PBST, each time for 3 min, and pat dry; add 100 μL / well of monoclonal 10A8 cell supernatant, and use 2 mouse tail blood diluted 1:1000 as the positive control, and use pCDNA3.1 cell supernatant as the negative control, each well is added with 100 μL, and incubated at 37°C for 1 h; wash 3 times with 0.1M PBST, each time for 3 min, and pat dry; use HRP-labeled secondary antibody (total IgG / IgG1 / IgG2a / IgG2b / IgG3 / IgM / IgKappa chain / IgLambda chain), antibody 1:5000 dilution (manufacturer: Jackson), 37°C incubation for 30 min; wash 3 times with 0.1M PBST, each time for 3 min, and pat dry; add TMB for color development for 5 min, use 2M H2SO4 to terminate color development, and measure the absorbance value at 450 nm.

[0090] The experimental results are as follows Figure 3As shown, the heavy chain of the 10A8 murine monoclonal antibody is IgG1, and the light chain is Kappa. The 10A8 murine monoclonal antibody was then purified from the culture supernatant of positive hybridoma cells as a positive control protein; the protein concentration was determined to be 0.6 mg / mL using the BCA method. Subsequent experiments were conducted using 10A8 hybridoma cells.

[0091] Step 2: Expand the culture of 10A8 hybridoma cells, lyse the cells with Trizol, and extract total RNA from the hybridoma cell lysate (Quick-RNA MicroPrep Kit). Separate the RNA by agarose gel electrophoresis, and then perform 5' RACE to obtain cDNA (Clontech SMARTer RACE 5' / 3' kit). Use this cDNA as a template for PCR amplification. MaxDNAPolymerase was used to extract cDNA from the variable region of the antibody, which was then sequenced by a sequencing company to obtain the amino acid sequences of the light chain and heavy chain variable regions of the 10A8 murine monoclonal antibody. SEQ ID No. 1 shows the amino acid sequence of the light chain variable region of the 10A8 murine monoclonal antibody, and SEQ ID No. 6 shows the amino acid sequence of the heavy chain variable region of the 10A8 murine monoclonal antibody. The antigenic determination complementary region (CDR) of the 10A8 murine monoclonal antibody amino acid sequence was labeled using the Kabat method. SEQ ID No. 5 shows the nucleotide sequence of the light chain variable region of the 10A8 murine monoclonal antibody, and SEQ ID No. 10 shows the nucleotide sequence of the heavy chain variable region of the 10A8 murine monoclonal antibody.

[0092] Example 4

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

[0094] Step 1: Construction of the recombinant vector

[0095] The nucleotide sequence of the variable region of the light chain of the anti-human Hu protein 10A8 murine monoclonal antibody (SEQ ID No. 5) was amplified, and the nucleotide sequence of the constant region of the human Kappa chain (SEQ ID No. 13) was amplified. The two fragments were ligated into the vector pcDNA3.1 using homologous recombination (manufacturer: TransGen, catalog number: CU201-02), labeled as pcDNA3.1-Hu-10A8-VL. The ligated recombinant plasmid was sent to Sangon Biotech for sequencing. The sequenced recombinant plasmid was then used for cell transfection to prepare antibodies.

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

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

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

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

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

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

[0102] The amplification procedure for both SEQ ID No. 5 and SEQ ID No. 13 is as follows: 95℃ pre-denaturation for 5 min ； 95℃ denaturation for 30 s, 62℃ annealing for 20 s, 72℃ extension for 20 s, 30 cycles; 72℃ re-extension for 3 min.

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

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

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

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

[0107] The procedure used for amplifying the sequence shown in SEQ ID No. 10 is as follows: 95°C pre-denaturation for 5 min ； 95°C denaturation for 30 s, 58°C annealing for 20 s, 72°C extension for 20 s, 30 cycles; 72°C re-extension for 3 min.

[0108] The primers used for amplifying 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 for amplifying the sequence shown in SEQ ID No. 14 is as follows: 95°C pre-denaturation for 5 min ； 95°C denaturation for 30 s, 64°C annealing for 20 s, 72°C extension for 30 s, 30 cycles; 72°C re-extension for 3 min.

[0112] Step two, expression and purification of protein

[0113] When the cell density is 4.5 million / mL, and the total cell volume is 100 mL, co-transfect 175 μg of plasmid at a ratio of heavy chain plasmid to light chain plasmid of 1:1, and the amount of PEI is three times the amount of plasmid, that is, 525 μg. Co-transfect 87.5 μg of each of the newly constructed pcDNA3.1-Hu-10A8-VL plasmid and pcDNA3.1-Hu-10A8-VH plasmid into Expi293F cells for transient expression (transfection reagent PEI, thermo), to obtain cells transfected with the anti-human Hu protein human-mouse chimeric monoclonal antibody 10A8 plasmid. The supernatant is collected after 3 days of transfection, and protein A filler is used for purification, and is labeled as Hu-10A8. The Hu-10A8 protein concentration is determined by the BCA method to be 0.4 mg / mL, as shown in FIG. 6. The Hu-10A8 human-mouse chimeric monoclonal antibody is successfully expressed and purified. Figure 4

[0114] Example 5

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

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

[0117] ​Using purified HuB-6his protein and HuD-6his protein to coat an ELISA plate respectively, 100 ng / well, 4°C coating overnight (coating solution is 25 mL carbonate buffer, pH 9.6); the next day, use PBST solution to wash 3 times, 3 min each time, and pat dry each time; add 2% BSA to fill the well and block, incubate at 37°C for 1 h; after incubation, wash 3 times with PBST, 3 min each time, and pat the plate to remove excess moisture each time; incubate the Hu-10A8 human-mouse chimeric antibody prepared in Example 4 and the 10A8 mouse-derived monoclonal antibody prepared in Example 1 as positive controls, and normal mouse serum as a negative control, with dilution ratios of 1:1, 1:3, 1:9, 1:27, 1:81, 1:243, 1:729, and 1:2187, respectively; add 100 μL of the above dilutions to each well, and incubate at 37°C for 1 h; wash 3 times with PBST, 3 min each time, and pat dry; dilute the goat anti-human / goat anti-mouse IgG-HRP secondary antibody at a ratio of 1:5000, add to the plate, and incubate at 37°C for 30 min; after incubation, wash 3 times with PBST, pat dry, add TMB for color development for 3 min, stop with 2M H2SO4, and measure the absorbance at 450 nm.

[0118] The experimental results are shown in Figure 5 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 the same as that of the unmodified 10A8 mouse-derived monoclonal antibody.

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

[0120] To verify whether the prepared human-mouse chimeric antibody has the ability to target and detect human HuB and HuD antigens, dot blotting was performed using the purified Hu-10A8 monoclonal antibody prepared in Example 3, and the test steps are as follows:

[0121] The protein distribution is shown in Figure 6As shown, wherein the quality control point (Ctrl) is an antibody against human Fab (manufacturer: sigma), with a concentration of 100 ng / μL; HuB is HuB-6his protein, and HuD is HuD-6his protein, diluted to a concentration of 50 ng / μL, 0.8 μL of the sample was taken using a micropipette and spotted on the NC membrane in the shape of a right trapezoid in a circular pattern, dried at 37°C for 30 min, and placed in a 12-well plate, and incubated with Hu-10A8 human-mouse chimeric monoclonal antibody (1:200 dilution) purified in Example 4, Hu antibody positive (i.e., both HuB and HuD antibodies are 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), and 10A8 mouse monoclonal antibody purified in Example 3 (1:200 dilution) at a volume of 300 ul / well, and incubated at room temperature for 30 min, washed with 1% NaCl for 4 min each time, for a total of 2 times; 2000-fold diluted AP-labeled goat anti-human / mouse IgG secondary antibody (manufacturer: jackson) was added to each well, and incubated at room temperature for 30 min, washed with 1% NaCl for 4 min each time, for a total of 2 times; after removing the washing solution in the wells, 50-fold diluted BCIP / NBT substrate was added at a volume of 300 μL / well, and incubated at room temperature in the dark for 4 min, the substrate dilution solution in the wells was discarded, and the test strip was rinsed with distilled water for 1-2 times, taken out, air-dried, or oven-dried at 37-45°C, and the color development was observed, and the results are shown in Figure 6

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

[0123] 3. Identification of the function of the anti-human Hu human-mouse chimeric monoclonal antibody by Western-Blot ​

[0124] The whole bacteria lysate of prokaryotic HuB and HuD before and after induction was prepared according to the construction of Example 1; 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 by RIPA lysis buffer, centrifuged to obtain the supernatant, and labeled as HuB-pCDNA3.1 and HuD-pCDNA3.1 and pCDNA3.1 for standby; the above proteins were subjected to gel electrophoresis, and after electrophoresis, wet transfer was performed at 300 mA for 90 min; 5% skim milk was blocked at room temperature for 1 h; Hu-10A8 human-mouse chimeric antibody (concentration: 0.4 mg / mL) diluted 1:2000 and commercial HuA / B / C / D antibody (manufacturer: Wuhan Sanying, product number: 13032-1-AP) were used to incubate the PVDF membrane after transfer, and incubated at 4°C overnight; the next day, TBST was washed 3 times, each for 5 min; HRP-labeled goat anti-human / rabbit secondary antibody (manufacturer: Jackson) was added and incubated at room temperature for 1 h; TBST was washed 3 times, each for 5 min; ECL chemiluminescence solution was added for color development and photography.

[0125] The results, as shown in Figure 7 Hu-10A8 human-mouse chimeric antibody can recognize HuB protein and HuD protein in overexpressing cells, and can also recognize HuB and HuD protein after prokaryotic induction expression, and has obvious humanization species advantage compared with commercial antibody, and can be used as a serum substitute to meet the long-term detection reagent demand.

[0126] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which are within the scope of the present application.

Claims

1. A human-mouse chimeric monoclonal antibody against human Hu protein, characterized in that, The structure includes heavy chains and light chains; The light chain includes a variable region and a constant region. The variable region includes complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID No. 2, the amino acid sequence of CDR2 is shown in SEQ ID No. 3, and the amino acid sequence of CDR3 is shown in SEQ ID No.

4. The 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 complementarity-determining regions CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID No. 7, the amino acid sequence of CDR2 is shown in SEQ ID No. 8, and the amino acid sequence of CDR3 is shown in SEQ ID No.

9. The heavy chain constant region is the 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 variable region of the light chain 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 constant region of the light chain 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. Recombinant cells expressing the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6.

9. The use of the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6 in the preparation of a reagent for the specific detection of human Hu autoantibodies.

10. The use of the human-mouse chimeric monoclonal antibody according to any one of claims 1 to 6 in the preparation of reagents for the diagnosis and / or prognosis of paraneoplastic syndromes of the nervous system.

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