Anti-mak16-iggl monoclonal subtype antibody and preparation method and application thereof

Abstract

The application discloses an anti-MAK16-IgG1 monoclonal subtype antibody and a preparation method and application thereof, and belongs to the technical field of immunological disease drugs. The application successfully prepares a high-concentration high-purity monoclonal antibody against MAK16 protein, which is a monoclonal IgG1 subtype antibody, namely anti-MAK16-IgG1 monoclonal subtype antibody; the titer is 160000; compared with RAW264.7 cells in a group stimulated and induced by IgG1+LPS, the expression amounts of IL-6 and IL-10 in RAW264.7 cells after 24h and 48h of stimulation and induction by the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the application and LPS are significantly reduced, and the difference is more obvious with the prolongation of time; the effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody is not less than 50 mu g / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunological disease drugs, in particular to an anti-MAK16-IgG1 monoclonal subtype antibody and a preparation method and application thereof. BACKGROUND

[0002] When MAK16 gene mutates, it will make Saccharomyces cerevisiae show temperature-sensitive growth properties, and plays a key role in the maturation process of 25S rRNA and 5.8S rRNA of Saccharomyces cerevisiae bacteria. Studies have shown that MAK16 protein plays an important role in the biosynthesis of 60S ribosome subunits and has extensive interactions with the rest of the proteins on the 60S ribosome subunit.

[0003] Interleukin 6 (IL-6) is a multifunctional cytokine that can exhibit pro-inflammatory and anti-inflammatory activities depending on the immune environment in the body. When the body is infected or tissue is damaged, IL-6 is rapidly produced and participates in host defense. On the other hand, excessive secretion of IL-6 can cause acute and chronic systemic inflammatory response and cytokine storm. The hexameric complex of IL-6 (IL-6 / IL-6R / gp130) executes various biological effects by activating different signal transduction mechanisms, including JAK / STAT3, Ras / MAPK, PI3K–PKB / Akt, etc., leading to chronic immune diseases such as cancer, rheumatoid arthritis, inflammatory bowel disease, etc. Studies have shown that in the pre-clinical manifestation of systemic lupus erythematosus (SLE), accompanied or preceded by the appearance of various autoantibodies, IL-6 levels can be detected in serum, and serum IL-6 levels are also elevated in active SLE patients, neuropsychiatric SLE, and high cardiovascular disease risk lupus patients.

[0004] Similar to the immune function of IL-6, interleukin 10 (IL-10) also has both anti-inflammatory and pro-inflammatory properties in immune responses: on the one hand, it can inhibit the expression and / or function of inflammatory cytokines such as IFN-γ, TNF, IL-1, IL-6, etc. in monocytes and T cells under certain conditions, on the other hand, it can also stimulate the proliferation and differentiation of autoreactive B cells to form plasma cells, thereby releasing immunoglobulins and other factors that promote immune inflammatory responses. Studies have shown that in lupus model mice, continuous administration of blocking anti-IL-10 antibodies at birth can delay the occurrence of autoantibodies and the appearance of symptoms such as proteinuria and glomerulonephritis.

[0005] Systemic lupus erythematosus (SLE) is a chronic diffuse connective tissue disease mainly caused by abnormal activation of the immune system attacking the body's own tissues, the specific cause of which is not clear, and the symptoms are very diverse, often including fever, light sensitivity, skin rash, lymph node enlargement, muscle and joint pain, headache, fatigue, etc. The disease can damage various organs of the body, and thus can cause various complications, including kidney damage, neuropsychiatric symptoms, etc.

[0006] The treatment of systemic lupus erythematosus usually relies on multiple therapies, including drug therapy, phototherapy and lifestyle changes, etc. Common drug therapies mainly include non-steroidal anti-inflammatory drugs, anti-malarial drugs, immunosuppressive drugs and corticosteroids, etc., and there is currently no complete cure. SUMMARY

[0007] The purpose of the present application is to provide an anti-MAK16-IgG1 monoclonal subtype antibody and its preparation method and application, so as to provide a candidate monoclonal antibody for treating immune diseases, enrich the types of drugs for treating or pre-preventing the onset of SLE, and contribute to the early conquest of autoimmune diseases.

[0008] To achieve the above-mentioned purpose, the present application provides a preparation method of an anti-MAK16-IgG1 monoclonal subtype antibody, comprising the following steps:

[0009] S1, preparing MAK16 protein as an antigen;

[0010] S2, immunizing mice with the antigen, preparing mouse polyclonal antisera, and detecting the titer;

[0011] S3, cell electrofusion and cell subcloning and detecting the titer;

[0012] S4, sequencing hybridoma cells;

[0013] S5, preparing mouse monoclonal antibodies, and obtaining anti-MAK16-IgG1 monoclonal subtype antibodies after purification.

[0014] Preferably, in S1, the gene sequence of MAK16 is subcloned into the prokaryotic expression vector pET-21a (+), and then the competent cells are transformed and the protein expression is induced, and after crude extraction of the protein, the protein is purified, and the purified protein is used as the antigen.

[0015] Preferably, in S3, the cells used for cell electrofusion are B cells of the mouse with the highest titer in S2 and non-secretory SP2 / 0 myeloma cells.

[0016] Preferably, the cell subcloning in S3 is performed twice, first, the well-growing hybridoma cells are collected to prepare a single cell suspension, and after 3 days of culture, single clones are selected, and cultured to the 8th day, and the wells with OD values consistent with the positive serum are selected for re-subcloning.

[0017] A method for preparing an anti-MAK16-IgG1 monoclonal subtype antibody as described above.

[0018] The anti-MAK16-IgG1 monoclonal subtype antibody as described above is used for preparing a drug for treating immune diseases.

[0019] Preferably, the immune diseases include rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus.

[0020] Therefore, the present application provides an anti-MAK16-IgG1 monoclonal subtype antibody, a preparation method and application thereof, and the specific technical effects are as follows.

[0021] (1) The present application successfully prepares a high-concentration and high-purity monoclonal antibody against MAK16 protein, which is a monoclonal IgG1 subtype antibody, namely, an anti-MAK16-IgG1 monoclonal subtype antibody, and the titer is 160000.

[0022] (2) Compared with the RAW264.7 cells induced by the mouse IgG1 type antiserum + LPS, the RAW264.7 cells induced by the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the present application + LPS for 24 h and 48 h have significantly reduced IL-6 and IL-10 expression amounts, and the difference is more obvious as the time is prolonged; the effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody is not less than 50 μg / mL.

[0023] The technical solutions of the present application will be further described in detail below with the aid of the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments of the present application will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative labor on the basis of these drawings.

[0025] Figure 1 is the SDS-PAGE detection result of the MAK16 protein in Example 1 of the present application;

[0026] Figure 2 is the titer detection result of the mouse polyclonal antiserum in the embodiment 1 of the present application;

[0027] Figure 3 is the ELISA detection result of the secondary subcloning cell antibody titer in the embodiment 1 of the present application, wherein a and b are the binding conditions of the hybridoma cell secreted antibody and antigen in each well; c is the Ig2a clone with relatively high expression amount; d is the Ig2b clone with relatively high expression amount;

[0028] Figure 4 is the SDS-PAGE and ELISA detection result of the monoclonal antibody in the embodiment 1 of the present application; wherein a is the SDS-PAGE detection result; b is the ELISA detection result;

[0029] Figure 5 is the SDS-PAGE and ELISA detection result of the anti-MAK16-IgG1 monoclonal subtype antibody after purification in the embodiment 1 of the present application; wherein a is the SDS-PAGE detection result; b is the ELISA detection result;

[0030] Figure 6 is the change of IL-6, IL-10 secretion amount after the anti-MAK16-IgG1 monoclonal subtype antibody acts for different time in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0031] Figure 7 is the change of IL-6, IL-10 gene expression level after the anti-MAK16-IgG1 monoclonal subtype antibody acts for different time in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0032] Figure 8 is the change of IL-6, IL-10 secretion amount after the antibody acts for 48h in different doses in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0033] Figure 9 is the change of IL-6, IL-10 gene expression level after the antibody acts for 48h in different doses in the embodiment 2 of the present application; wherein a is IL-6; b is IL-10; * represents P<0.05;

[0034] Figure 10 is the p-STAT3 / STAT3, p-AKT / AKT ratio and Western blot representative graph after the antibody acts for different time in the embodiment 3 of the present application; wherein a is the p-STAT3 / STAT3 ratio; b is the p-AKT / AKT ratio; c is the Western blot representative graph;

[0035] Figure 11 Figure 8 is a representative Western blot of p-STAT3 / STAT3, p-AKT / AKT ratio and Western blot after 40 min of the effect of different doses of antibodies in Example 3; wherein a is the p-STAT3 / STAT3 ratio; b is the p-AKT / AKT ratio; c is a representative Western blot. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0037] In order to make the purpose, technical solutions and advantages of the present application more clear, thorough and complete, the technical solutions of the present application are described clearly and completely below by means of the accompanying drawings and examples. The following detailed description is a description of the examples, which aims to provide further detailed description of the present application. Unless otherwise specified, all technical terms used in the present application have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0038] The instruments and reagents used in the examples were obtained through commercial channels; wherein the serum samples of 30 patients in the experimental group with high levels of anti-MAK16 (chip method and ELISA method detection values are greater than the corresponding critical value) and 15 patients in the disease control group with low levels of anti-MAK16 (chip method and ELISA method detection values are less than the corresponding critical value) SLE patient serum samples were collected from the clinical biological samples collected by the First Affiliated Hospital of Anhui Medical University, 20 serum samples of the healthy control group were collected from the biological samples of the healthy examination population collected by the First Affiliated Hospital of Anhui Medical University, the patients in the experimental group and the healthy control group and the disease control group were roughly matched in gender and age; the RAW264.7 cells of mice were derived from the American Type Culture Collection (ATCC).

[0039] The experiments in the examples were approved by the Ethics Committee of Anhui Medical University (Ethics Approval No: 20180078) before implementation, and all patients and control groups have signed the informed consent form before participating in the study.

[0040] The method steps not described in detail in the examples are conventional technical means in the art.

[0041] Example 1

[0042] The anti-MAK16-IgG1 monoclonal subtype antibody was prepared, and the specific steps were as follows:

[0043] (1) Preparation of antigen.

[0044] 1) The amino acid sequence information of the target gene MAK16 retrieved from UniProt (as shown in SEQ ID NO. 1) was sent to a company for codon optimization and gene synthesis. The synthesis product was subcloned into a prokaryotic expression vector pET-21a(+), and after gene sequencing confirmed that the sequence in the inserted vector was correct, the recombinant prokaryotic expression vector pET-21a(+)-MAK16 was obtained.

[0045] SEQ ID NO. 1:

[0046] MQSDDVIWDTLGNKQFCSFKIRTKTQSFCRNEYSLTGLCNRSSCPLANSQYAT

[0047] IKEEKGQCYLYMKVIERAAFPRRLWERVRLSKNYEKALEQIDENLIYWPRFIR

[0048] HKCKQRFTKITQYLIRIRKLTLKRQRKLVPLSKKVERREKRREEKALIAAQLD

[0049] NAIEKELLERLKQDTYGDIYNFPIHAFDKALEQQEAESDSSDTEEKDDDDDDE

[0050] EDVGKREFVEDGEVDESDISDFEDMDKLDASSDEDQDGKSSSEEEEEKALSA

[0051] KHKGKMPLRGPLQRKRVYVEIEYEQETEPVAKAKTT

[0052] 2) The recombinant prokaryotic expression vector pET-21a(+)-MAK16 was transformed into BL21 competent cells, and 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used for induction at 37°C for 5 h, and the induction was expressed as an inclusion body.

[0053] 3) The protein was subjected to crude extraction, and the expressed protein was purified by nickel column. The purified sample was dialyzed into phosphate buffered saline (PBS) and concentrated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect the expression results of MAK16 protein. The results are shown in FIG. 1. Figure 1The target antigen, MAK16 protein, was obtained as shown.

[0054] (2) Antigen immunization of mice. All mice were raised in a barrier system, using sterilized pellet feed and autoclaved drinking water for feeding. Five BALB / c mice (SPF level) were ear-tagged and immunized with purified MAK16 recombinant protein according to the immunization schedule (three immunizations, first blood collection, fourth immunization, second blood collection, and final booster).

[0055] (3) Preparation of mouse polyclonal antiserum samples:

[0056] 1) After the third and fourth immunizations, blood was collected from the tail vein of the mice, about 20 μL.

[0057] 2) The blood samples were placed in a 4°C refrigerator and allowed to stand overnight.

[0058] 3) Then, centrifugation was performed at 4°C and 10,000 g for 10 min, and the yellow serum layer was transferred to a new test tube and stored at -20°C.

[0059] 4) The positive serum obtained above was diluted at ratios of 1:1000, 1:5000, 1:20000, 1:40000, 1:80000, and 1:160000, and PBS and negative serum controls were set, and then the serum titers were detected by ELISA.

[0060] (4) ELISA detection of antiserum titers:

[0061] 1) The purified MAK16 recombinant protein was diluted with PBS, and the protein concentration was adjusted to 5 mg / mL.

[0062] 2) 100 μL of the 5 mg / mL antigen sample was added to each well of a 96-well plate, and the plate was coated at 4°C overnight.

[0063] 3) Washing was performed three times with PBST (PBS buffer containing 0.5% Tween 20).

[0064] 4) Blocking was performed at room temperature for 1 h using 5% BSA.

[0065] 5) Step 3) was repeated three times.

[0066] 6) 100 μL of the diluted positive serum (1:1000, 1:5000, 1:20000, 1:40000, 1:80000, and 1:160000) and PBS and negative serum controls were added to the enzyme-labeled plate coated with the corresponding antigen.

[0067] 7) Incubation was performed at 37°C for 1 h.

[0068] 8) Repeat step 3) washing 3 times.

[0069] 9) Add 100 μL 1:20000 diluted HRP labeled anti-mouse IgG per well.

[0070] 10) Incubate at 37°C for 1 h.

[0071] 11) Repeat step 3) washing 3 times.

[0072] 12) Add 100 μL TMB color developing solution per well, and develop color for 15 min.

[0073] 13) Add 50 μL stop solution to stop the color developing reaction, and use an enzyme label meter to detect.

[0074] The results are shown in Table 1. Figure 2 As shown in Table 1, the B4 mouse has a better antibody titer for the target antigen, and therefore, the B4 mouse is selected for challenge immunization, and its immune B cells are taken for cell electrofusion and titer detection.

[0075] (5) Cell electrofusion:

[0076] 1) The mouse with the highest titer is sacrificed by cervical dislocation, and the mouse spleen is obtained under sterile conditions to prepare a B cell single cell suspension, which is mixed with non-secretory SP2 / 0 myeloma cells at a ratio of 1:1, and Bio-rad Gene electroporation system is used for cell fusion.

[0077] 2) After electrofusion, all cells are immediately suspended in complete culture medium (DMEM, 20% FBS and HAT), and inoculated into a 96-well plate.

[0078] 3) About 10 days after fusion, the medium is changed to HT medium, and after two days of culture, 100 μL of supernatant is taken for ELISA detection, and the corresponding wells with OD values consistent with positive serum (1:1000) are selected for subcloning.

[0079] (6) Cell subcloning:

[0080] 1) Collect the hybridoma cell strain in good growth condition to prepare a single cell suspension.

[0081] 2) Count using a hemocytometer, and take 100 cells, which are diluted to 10 mL.

[0082] 3) Take a 96-well plate, and add 100 μL of cell suspension per well, and finally supplement the volume to 200 μL.

[0083] 4) Place in a 37°C incubator, and label.

[0084] 5) After 3 days of continuous culture, single clones are selected under microscope.

[0085] 6) Culture to day 7, replace fresh medium.

[0086] 7) Culture to day 8, detect cell supernatant using ELISA method, select wells with OD value consistent with positive serum (1:1000) for secondary subcloning.

[0087] After cell electrofusion and secondary subcloning, cells are plated in 96-well plates, and hybridoma cells in each well secrete antibodies, and the antigen binding condition is shown in Figure 3 A and Figure 3 B, and Ig2a and Ig2b clones with relatively high expression are selected as shown in Figure 3 C and Figure 3 D. At this time, the cells in each well can be polyclonal or monoclonal, and further microscopy is performed to confirm monoclonal cells, and wells containing only one clone are selected for subsequent experiments.

[0088] (7) Preparation and purification of mouse monoclonal antibodies. The hybridoma cells determined by subcloning titer detection are added to 1.5 mL centrifuge tubes under sterile conditions, 250g / min, 4°C centrifugation for 10 min, and the supernatant is removed. Wash 3 times with 1xPBS, resuspend the hybridoma cells with 500 μL 1xPBS, inject the cells into the abdomen of mice with a 1 mL syringe, and wait for 7-10 days to collect ascites. The antibody is purified by ammonium sulfate precipitation and Protein G affinity chromatography, and the antibody titer is detected by SDS-PAGE and ELISA. 6

[0089] The SDS-PAGE diagram is shown in Figure 4 a, and the ELISA detection result is shown in Figure 4 b, which shows that a high concentration of IgG2 subtype antibody is obtained.

[0090] (8) Hybridoma cell gene sequencing (1-2-d clone strain). The variable region sequences of VH and VL are amplified according to the standard operating procedures of the laboratory, the VH and VL variable regions of the 1-2-d clone strain are respectively cloned into T vectors using standard molecular cloning methods, blue-white spot screening is performed, the spot is shaken, and gene sequencing is performed.

[0091] ​The variable region sequences of 1-2-d clone strains VH and VL were amplified according to the standard operation procedure, the VH and VL variable regions were cloned into T vectors respectively using standard molecular cloning method, blue-white spot screening was performed, the spots were shaken, sequencing was performed, the nucleotide sequence of heavy chain 1 was shown as SEQ ID NO. 2, the nucleotide sequence of light chain 1 was shown as SEQ ID NO. 3, and the nucleotide sequence of light chain 2 was shown as SEQ ID NO. 4. The sequencing results showed that the two light chains only had one amino acid difference in the CDR1 region (the italicized and underlined font sequentially identified CDR1, CDR2, CDR3; wherein the CDR1 in the light chain was shown by italicized and underlined bold font as the difference nucleotide).

[0092] SEQ ID NO. 2:

[0093]

[0094] SEQ ID NO. 3:

[0095]

[0096]

[0097] SEQ ID NO. 4:

[0098]

[0099] (9) Expression and purification of target antibody. According to the sequencing results of the previous hybridoma, a mouse IgG1 antibody expression vector was constructed, and an endotoxin-free plasmid was prepared for standby. The LVTransm transfection reagent and antibody expression vector were taken out and thawed at room temperature, and then mixed completely by blowing with a micropipette. Phosphate buffered saline (PBS) was taken out and balanced to room temperature. 2 mL of PBS was taken into different wells of a reaction plate using a micropipette, 130 μg of antibody expression vector was added to each well, and the micropipette was blown to mix thoroughly. Then 400 μL of LVTransm was added to each well, and the micropipette was blown again to mix thoroughly. The DNA / LVTransm complex was added to a culture bottle containing 50 mL of 293F-SVP16 cells, and the culture bottle was gently shaken to mix the complex and the cells thoroughly. The culture bottle was placed in a 37°C incubator containing 5% CO2, and cultured at a shaking speed of 130 rpm for 6-8 h. Then 50 mL of fresh FreeStyle 293 Expression Medium was added to the culture bottle, and the cells were cultured at a shaking speed of 130 rpm for 6-8 h. TM293Expression Medium, and the cell culture flask was put back into the incubator for continued shaking culture. After 7 days of continuous culture, the medium supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred into a sterile centrifuge tube. The antibody was purified using a Protein G column and subjected to ELISA detection.

[0100] The results, as shown in Figure 5 indicate that a higher concentration of IgG1 subtype antibody was obtained.

[0101] Example 2

[0102] The effect of the anti-MAK16-IgG1 monoclonal subtype antibody prepared in Example 1 on IL-6 and IL-10 was investigated, as follows:

[0103] The mouse monocyte macrophage leukemia cell RAW264.7 purchased from ATCC was placed in DMEM medium containing 10% FBS and cultured in a cell incubator at 37°C in a 5% CO2 environment.

[0104] In the first stage, the RAW264.7 cells after overnight culture were adjusted to a cell concentration of 5 x 10 4 cells / mL, and the experiment was divided into 4 groups (RAW264.7 group; RAW264.7 + 1 μg / mL LPS group; RAW264.7 + 100 μg / mL mouse IgG1 + 1 μg / mL LPS group; RAW264.7 + 100 μg / mL anti-MAK16-IgG1 + 1 μg / mL LPS group). LPS and antibodies were added, respectively, and each group had 3 replicate wells. After 12 h, 24 h, and 48 h of continued culture, the culture supernatant was taken for ELISA detection.

[0105] In the second stage, the RAW264.7 cells after overnight culture were adjusted to a cell concentration of 5 x 10 4cells / mL, and the LPS and different doses of antibodies were added respectively, and each group had 3 replicate wells. After a period of time, the culture supernatant was taken for ELISA detection.

[0106] The RAW264.7 cells were cultured overnight, and the cell concentration was adjusted to 5 x 105cells / mL. 4 After the mixed culture stimulation and induction of the LPS, the antibodies, and the cells in different groups in the two stages of the above ELISA detection process, the cell RNA was extracted, and the expression levels of IL-6 and IL-10 genes were detected by RT-qPCR, and each group was repeated three times.

[0107] The detection results of the first-stage ELISA and RT-qPCR detection are shown in Tables 1-2 and Figure 6-7 As shown in Tables 1-2 and Figs. 1-2, after the RAW264.7 cells were stimulated and induced by LPS (1 μg / mL), LPS (1 μg / mL) + mouse IgG1 (100 μg / mL), or LPS (1 μg / mL) + anti-MAK16-IgG1 (100 μg / mL) for 12 h, 24 h, and 48 h, the expression levels of IL-6 and IL-10 increased with the extension of time. However, after the stimulation and induction for 24 h and 48 h, the expression levels of IL-6 and IL-10 in the anti-MAK16-IgG1 stimulation and induction group were reduced relative to the same group (P < 0.05), and the difference was more obvious with the extension of time.

[0108] Table 1 ELISA detection of the secretion of IL-6 and IL-10 after the action of the antibodies for different time

[0109]

[0110] Table 2 RT-qPCR detection of the expression levels of IL-6 and IL-10 genes after the action of the antibodies for different time

[0111]

[0112] The detection results of the second stage ELISA and RT-qPCR detection are shown in Tables 3-4 and Figure 8-9 As shown in Tables 3-4 and 5, after RAW264.7 cells were co-stimulated by LPS (1 μg / mL) and mouse IgG1 or anti-MAK16-IgG1 at concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL for 48 h, the expression levels of IL-6 and IL-10 showed no significant change; with the increase of the dose of anti-MAK16-IgG1, the expression levels of IL-6 and IL-10 showed a decreasing trend; after being stimulated by anti-MAK16-IgG1 at doses of 50 μg / mL and 100 μg / mL, the expression levels of IL-6 and IL-10 were lower than those of the control group at the same dose (P<0.05).

[0113] Table 3 ELISA detection of IL-6 and IL-10 secretion after 48 h of action of different doses of antibodies

[0114]

[0115]

[0116] Table 4 RT-qPCR detection of the expression levels of IL-6 and IL-10 genes after 48 h of action of different doses of antibodies

[0117] Group IL-6 IL-10 RAW264.7 1±0.06 1±0.06 RAW264.7+LPS 9.12±0.4 7.73±0.38 RAW264.7+mouse IgGl (25 μg / mL)+LPS 10.28±0.46 7.59±0.66 RAW264.7+mouse IgGl (50 μg / mL)+LPS 10.67±0.38 7.86±0.29 RAW264.7+mouse IgGl (100 μg / mL)+LPS 9.84±0.36 7.52±0.37 RAW264.7+anti-MAK16-IgGl (25 μg / mL)+LPS 7.79±0.34 6.85±0.29 RAW264.7+anti-MAK16-IgGl (50 μg / mL)+LPS 5.17±0.34 5.53±0.20 RAW264.7+anti-MAK16-IgGl (100 μg / mL)+LPS 2.82±0.19 4.78±0.29

[0118] Example 3

[0119] The effect of anti-MAK16-IgG1 monoclonal subtype antibody prepared in Example 1 on the levels of p-STAT3 / STAT3 and p-AKT / AKT in cells was investigated by Western blot, and each group was set the same as the first stage of Example 2. The protein concentrations of each test sample are shown in Table 5.

[0120] Table 5 Protein concentrations of each test sample

[0121]

[0122]

[0123] The results are shown in Tables 6 and 7. Figure 10As shown in the experimental data, after processing and statistical analysis, it was found that the expression of STAT3 and AKT in RAW264.7 cells did not change significantly after being stimulated by different antibodies and LPS (1 μg / mL) for different times. The expression of p-STAT3 and p-AKT reached a high value in the anti-MAK16-IgG1 (100 μg / mL) group (experimental group) and the mouse IgG1 (100 μg / mL) control group after being stimulated for 40 min, and then the expression decreased. The expression of p-STAT3 and p-AKT in the experimental group was lower than that in the mouse IgG1 control group (P < 0.05) at different stimulation times, but there was no difference between the LPS (1 μg / mL) control group and the mouse IgG1 (100 μg / mL) + LPS (1 μg / mL) control group (Table 6, Figure 10 ) after being stimulated by different doses of different antibodies and LPS for 40 min. The expression of STAT3 and AKT did not change significantly, and the expression of p-STAT3 and p-AKT in the experimental group decreased with the increase of the dose of anti-MAK16-IgG1. When the antibody with the highest dose was used, the expression of p-STAT3 and p-AKT in the experimental group was lower than that in the mouse IgG1 control group (P < 0.05) (Table 7, Figure 11 ).

[0124] Table 6 p-STAT3 / STAT3, p-AKT / AKT ratio after different antibodies act for different times

[0125]

[0126]

[0127] Table 7 p-STAT3 / STAT3, p-AKT / AKT ratio after different doses of antibodies act for 40 min

[0128] Group p-STAT3 / STAT3 p-AKT / AKT RAW264.7 0.266±0.039 0.291±0.043 RAW264.7+LPS 0.801±0.093 0.787±0.071 RAW264.7+mouse IgGl (25 μg / mL)+LPS 0.709±0.076 0.835±0.089 RAW264.7+mouse IgGl (50 μg / mL)+LPS 0.782±0.083 0.795±0.088 RAW264.7+mouse IgGl (100 μg / mL)+LPS 0.918±0.097 0.844±0.087 RAW264.7+anti-MAK16-IgGl (25 μg / mL)+LPS 0.680±0.073 0.860±0.097 RAW264.7+anti-MAK16-IgGl (50 μg / mL)+LPS 0.528±0.068 0.808±0.069 RAW264.7+anti-MAK16-IgGl (100 μg / mL)+LPS 0.375±0.048 0.585±0.073

[0129] Therefore, the anti-MAK16 protein monoclonal antibody with high concentration and high purity is successfully prepared, which is a monoclonal IgG1 subtype antibody, anti-MAK16-IgG1 monoclonal subtype antibody. The titer is 160000. Compared with the RAW264.7 cells stimulated by IgG1 + LPS, the expression of IL-6 and IL-10 in the RAW264.7 cells stimulated by the anti-MAK16-IgG1 monoclonal subtype antibody prepared by the present application and LPS for 24 h and 48 h is significantly reduced, and the difference becomes more obvious with the extension of time. The effective dose of the anti-MAK16-IgG1 monoclonal subtype antibody is not less than 50 μg / mL.

[0130] Finally, it should be noted that the above examples are intended to illustrate the technical solutions of the present application but not to limit the same. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still make modifications or equivalent replacements to the technical solutions of the present application, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. An anti-MAK16-IgGl monoclonal subtype antibody, characterized in that: The anti-MAK16-IgG1 monoclonal subtype antibody is composed of a heavy chain and a light chain, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 2, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO.

3. The anti-MAK16-IgG1 monoclonal subtype antibody is composed of a heavy chain and a light chain, the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO. 2, and the nucleotide sequence of the light chain variable region is shown as SEQ ID NO. 3.

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