WO2023165230A1 - 一种鼠抗mcr-1/mcr-2蛋白杂交瘤细胞株，单克隆抗体及应用 - Google Patents

一种鼠抗mcr-1/mcr-2蛋白杂交瘤细胞株，单克隆抗体及应用 Download PDF

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WO2023165230A1
WO2023165230A1 PCT/CN2022/141244 CN2022141244W WO2023165230A1 WO 2023165230 A1 WO2023165230 A1 WO 2023165230A1 CN 2022141244 W CN2022141244 W CN 2022141244W WO 2023165230 A1 WO2023165230 A1 WO 2023165230A1
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mcr
antibody
seq
protein
variable region
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French (fr)
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李可可
苑庆华
何永胜
陈晓玲
夏斌
王亚苗
孔迪
王思怡
杨彦霖
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Genobio Pharmaceutical Co Ltd
Tianjin Era Biology Technology Co Ltd
Tianjin Era Biology Techology Co Ltd
Beijing Gold Mountainriver Tech Development Co Ltd
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Genobio Pharmaceutical Co Ltd
Tianjin Era Biology Technology Co Ltd
Tianjin Era Biology Techology Co Ltd
Beijing Gold Mountainriver Tech Development Co Ltd
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Priority to EP22871086.9A priority Critical patent/EP4265717B1/en
Publication of WO2023165230A1 publication Critical patent/WO2023165230A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9129—Transferases for other substituted phosphate groups (2.7.8)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/44—Multiple drug resistance

Definitions

  • the invention belongs to the technical field of antibody preparation, and in particular relates to a mouse anti-MCR-1 and MCR-2 protein hybridoma cell line, a monoclonal antibody and its application.
  • Polymyxin is currently the last choice for clinical treatment of multidrug-resistant and pan-drug-resistant Gram-negative bacterial infections. Due to the increase in clinical use of polymyxin in recent years, the number of drug-resistant strains has also increased year by year. Although the current bacterial resistance to polymyxin is not strong, it may lead to the point where there is no cure. How to effectively curb the spread of drug resistance has become a global public health issue.
  • MCR genes that mediate the addition of lipid A phosphoethanolamine (PEtn) were found in a variety of plasmids, and can spread horizontally in different bacteria through plasmids, and can even co-exist with other drug resistance genes in The same plasmid can produce multiple drug resistance mechanisms after expression.
  • Ptn lipid A phosphoethanolamine
  • the present invention provides a mouse anti-MCR-1, MCR-2 protein hybridoma cell line, monoclonal antibody and application.
  • the technical solution adopted in the present invention is: a mouse anti-MCR-1/MCR-2 protein hybridoma cell line, named 2DG5, with a preservation number of CGMCC No. 23034; or named 1IB10, with a preservation number of CGMCC No. 23032.
  • Mouse anti-MCR-1/MCR-2 protein antibody including light chain variable region and heavy chain variable region
  • the light chain variable region includes CDRL1, SEQ ID NO:2 as shown in SEQ ID NO:1 CDRL2 shown and CDRL3 shown in SEQ ID NO:3
  • the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5, and CDRH2 shown in SEQ ID NO:6 CDRH3;
  • the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and CDRL3 shown in SEQ ID NO:13, and the heavy chain variable region includes CDRL3 shown in SEQ ID NO: CDRH1 shown in :14, CDRH2 shown in SEQ ID NO:15 and CDRH3 shown in SEQ ID NO:16;
  • amino acid sequence of the light chain variable region of the antibody 2DG5 is shown in SEQ ID NO: 7
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9;
  • amino acid sequence of the light chain variable region of antibody 1IB10 is shown in SEQ ID NO: 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19;
  • the antibody 2DG5 is produced by a mouse anti-MCR-1/MCR-2 protein hybridoma cell line with deposit number CGMCCNo.23034;
  • the antibody 1IB10 is produced by the mouse anti-MCR-1/MCR-2 protein hybridoma cell line with the preservation number CGMCC No. 23032.
  • a nucleic acid molecule comprising nucleotides encoding a mouse anti-MCR-1/MCR-2 protein antibody.
  • the nucleic acid molecule encodes the nucleotide sequence of the light chain variable region of antibody 2DG5 as shown in SEQ ID NO: 8, and the nucleic acid molecule encodes the nucleotide sequence of the heavy chain variable region of antibody 2DG5 as shown in SEQ ID NO: 10 shown;
  • nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1IB10 is shown in SEQ ID NO: 18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule encoding antibody 1IB10 is shown in SEQ ID NO: 20;
  • mouse anti-MCR-1/MCR-2 protein antibody in preparation of reagents for detecting MCR-1 or MCR-2 protein.
  • the mouse anti-MCR-1/MCR-2 protein antibody is used to prepare an in vitro diagnostic kit or a microfluidic chip, and the in vitro diagnostic kit is a colloidal gold immune kit, a chemiluminescence kit, a radioimmunoassay kit, Enzyme-linked immunoassay kit or fluorescent immunoassay kit.
  • antibody 2DG5 is a coating antibody
  • antibody 1IB10 is a gold-labeled antibody
  • antibody 1IB10 is a coating antibody
  • antibody 2DG5 is a gold-labeled antibody
  • the present invention provides two kinds of mouse anti-MCR-1/MCR-2 protein hybridoma cell lines, which can produce two kinds of mouse anti-MCR-1/MCR-2 protein antibodies respectively.
  • Antibodies can react specifically with MCR-1 protein or MCR-2 protein; through systematic evaluation, including the evaluation of antibody subtype and titer, kit sensitivity, specificity and stability, mouse anti-MCR-1/
  • the MCR-2 protein monoclonal antibody has good performance in all aspects, and the titer has reached more than 1:1280000, so it is suitable as an immunodiagnostic reagent for the preparation of an in vitro diagnostic kit for detecting polymyxin-resistant strains.
  • Fig. 1 is the electrophoresis diagram of mouse anti-MCR-1/MCR-2 protein protein
  • Fig. 2 is the protein electrophoresis diagram of mouse anti-MCR-1/MCR-2 protein antibody
  • Fig. 3 Detection results of mouse anti-MCR-1/MCR-2 protein detection card by colloidal gold method.
  • Biological material 2DG5, the preservation date is September 24, 2021, the preservation unit is the General Microbiology Center (CGMCC), and the preservation number is CGMCC No.23034;
  • CGMCC General Microbiology Center
  • Biological material 1IB10
  • the preservation date is September 24, 2021
  • the preservation unit is the General Microbiology Center (CGMCC) of the China Committee for the Collection of Microorganisms
  • the preservation number is CGMCC No.23032.
  • the invention relates to a mouse anti-MCR-1/MCR-2 protein hybridoma cell strain, the biological material is named 2DG5, which belongs to the hybridoma cell, and its preservation number is CGMCC No.23034; the preservation place is common microorganisms of China Microorganism Culture Preservation Management Committee Center, whose deposit date is September 24, 2021, was detected as alive.
  • the biological material is named 1IB10, which belongs to hybridoma cells, and its preservation number is CGMCC No.23032; the preservation place is Common Microorganisms of China Microbiological Culture Collection Management Committee Center, whose deposit date is September 24, 2021, was detected as alive.
  • the antibody 2DG5 produced by the mouse anti-MCR-1/MCR-2 protein hybridoma cell line includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes CDRL1 as shown in SEQ ID NO: 1, CDRL2 shown in SEQ ID NO:2 and CDRL3 shown in SEQ ID NO:3, the heavy chain variable region includes CDRH1 shown in SEQ ID NO:4, CDRH2 and SEQ ID NO shown in SEQ ID NO:5 : CDRH3 shown in 6;
  • amino acid sequence of the light chain variable region of antibody 2DG5 is shown in SEQ ID NO: 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9;
  • nucleotide sequence encoding the light chain variable region of antibody 2DG5 is shown in SEQ ID NO: 8, and the nucleotide sequence encoding the heavy chain variable region of antibody 2DG5 is shown in SEQ ID NO: 10;
  • the antibody 1IB10 produced by the mouse anti-MCR-1/MCR-2 protein hybridoma cell line the light chain variable region includes CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and SEQ ID NO CDRL3 shown in: 13, the heavy chain variable region includes CDRH1 shown in SEQ ID NO: 14, CDRH2 shown in SEQ ID NO: 15 and CDRH3 shown in SEQ ID NO: 16;
  • amino acid sequence of the light chain variable region of antibody 1IB10 is shown in SEQ ID NO: 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 19;
  • the nucleotide sequence encoding the light chain variable region of antibody 1IB10 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the heavy chain variable region of antibody 1IB10 is shown in SEQ ID NO: 20;
  • Mouse anti-MCR-1/MCR-2 protein monoclonal antibody passed the systematic evaluation, including the evaluation of antibody subtype and titer, kit sensitivity, specificity and stability; MCR-1 protein and MCR-2 protein were compared Similarly, the mouse anti-MCR-1/MCR-2 protein monoclonal antibody prepared by the present invention can specifically recognize both the MCR-1 protein and the MCR-2 protein, and has better performance in all aspects, thereby It is suitable as an immunodiagnostic reagent for the preparation of in vitro diagnostic kits.
  • colloidal gold immunoassay kit chemiluminescence kit, radioimmunoassay kit, enzyme-linked immunoassay kit or fluorescent immunoassay kit, or as a microfluidic chip; the kit can detect MCR-1, MCR-2 protein.
  • the two antibodies involved in this scheme are especially suitable for the formation of double-antibody sandwich immunocolloidal gold test strips, in which antibody 2DG5 is a coated antibody, antibody 1IB10 is a gold-labeled antibody, and the prepared colloidal gold test strips are more sensitive. , at the same time, antibody 2DG5 can also be used as a gold-labeled antibody, and antibody 1IB10 can be used as a coating antibody.
  • the purified MCR-1 protein was used to immunize female Balb/c mice about 6 weeks old for antibody preparation. According to the immunization dose, they were divided into 2 groups with 5 mice in each group; calculated according to the antigen content, the immunization dose of the first group was 25ug/mouse, the immunization dose of the second group is 50ug/mouse, for the first immunization, take an appropriate amount of MCR-1 protein and dilute it to 500ul with normal saline, add an equal amount of Freund's complete adjuvant to 500ul to emulsify evenly, and subcutaneously inject multiple points to immunize mice; Two weeks later, take the same dose for the second immunization.
  • the adjuvant was replaced by Freund's incomplete adjuvant, and the mice were immunized by intraperitoneal injection, and an additional immunization was given two weeks later, which was also an intraperitoneal injection to immunize the mice.
  • Blood was collected from the tail of the mouse, and the serum titer of the mouse was determined by ELISA.
  • the specific steps are: MCR-1 protein 0.2ug/ml, 100ul/well, coat the ELISA plate overnight at 4°C, spin dry, and wash 3 times with PBST. 5% skimmed milk powder, 200ul/well, sealed at 37°C for 2h.
  • the blood was collected from the tail of the mouse, centrifuged at 3000 rpm, and the serum was collected, starting from 1:1000 and diluted to 1:512000 with PBS, and set aside.
  • Spin dry, wash 3 times with PBST add primary antibody diluted in PBS from 1:1000 times, 100ul/well, 37 degrees, 1h.
  • Spin dry, wash 3 times with PBST add goat anti-mouse secondary antibody diluted 1:10000 times in PBS, 100ul/well, 37 degrees, 45min.
  • mice were boosted with the same amount of inoculation as the previous immunization, without adjuvant, and injected intraperitoneally.
  • Prepare the feeder layer cells one day before the fusion, take a 6-8 week-old mouse Balb/c, take the eyeballs and let them die by dislocation of the cervical spine after bloodletting, put them in 75% alcohol for 5 minutes, fix them on the plate, and keep them sterile in an ultra-clean table Cut the abdominal skin.
  • Use a sterile syringe to draw 10ml of the HAT selection culture solution and inject it into the abdominal cavity of the mouse, gently rub the abdomen with an alcohol cotton ball, and withdraw the culture medium.
  • HAT culture solution Add 40ml of HAT culture solution, spread into four 96-well cell culture plates, 100 ⁇ L/well, culture in 37°C, 5% CO 2 cell culture incubator.
  • Myeloma cells (Sp2/0 cells) were revived one week before fusion, cultured in PRMI-1640 medium containing 10% fetal calf serum, and subcultured in a 37°C, 5% CO 2 incubator. Collect the cells in the logarithmic growth phase into a centrifuge tube, count the cells, and dilute the cells to 10 7 cells/ml for later use.
  • Gently blow and blow the splenocyte suspension in the plate transfer to a 50ml centrifuge tube, centrifuge at 1000r/min for 5min, collect the splenocytes, and count them for later use.
  • the blue cap bottle is filled with 37°C hot water, add the preheated 1ml DMSO/PEG dropwise into the fusion tube within 1min, first slowly and then quickly, and gently rotate the centrifuge tube while adding.
  • the cell plate After 7 days, the cell plate will be half-replaced with fresh HAT medium, and fully replaced with HT medium after 10 days.
  • Subcloning the positive cells in the 96-well plate using the limiting dilution method first prepare the feeder layer cells according to the above method, take the hybridoma cells to be cloned for cell counting, and dilute the cells to 5-8 cells/cell with HT medium ml, add 100 ⁇ L/well to the 96-well cell plate that has been plated with feeder cells, one 96-well cell plate for each hybridoma cell clone, and culture in a 37°C, 5% CO 2 cell incubator.
  • the expansion culture can be carried out, and the strains can be determined and frozen.
  • the hybridoma cells that were positively detected and determined to be strained were expanded and cultured and frozen. The specific process is as follows: the vigorously growing hybridoma cells in good condition were gently blown off the cell bottle with anti-blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant was discarded.
  • cryopreservation solution containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, 10% DMSO
  • mice Female Balb/c mice aged 10-12 weeks were injected intraperitoneally with sterile liquid paraffin, 0.5 mL/mouse, and 7 days later, hybridoma cells cultured to logarithmic phase were injected intraperitoneally, 5 ⁇ 10 6 cells/mouse. Pay attention to observation every day for about 7-10 days. After the abdomen of the mice has obvious bulge, disinfect the skin of the lower abdomen with 75% alcohol cotton balls, puncture the abdominal cavity with a 16-gauge needle, and collect ascites. After ascites regenerates and accumulates, collect again. The collected ascites was centrifuged at 3000r/min for 10min, and the middle clarified part was taken, filtered with filter paper, aliquoted, and stored at -70°C.
  • the subclass identification of monoclonal antibody was carried out by the method of capture ELISA, as follows: After diluting the monoclonal antibody subclass identification reagent at 1:1000, add it to the enzyme-labeled well, 100 ⁇ L/well, and incubate at 37 ° C for 1 h; Wash three times with PBST and pat dry; dilute antibody 1:1000 times and add sample, 100 ⁇ L/well, incubate at 37°C for 1 h; wash three times with PBST, pat dry; HRP enzyme-labeled goat anti-mouse IgG secondary antibody diluted 1:10000 and add Sample, 100 ⁇ L/well, incubate at room temperature for 30 minutes; develop color for 10-20 minutes.
  • the subtype of the monoclonal antibody was defined as the OD 450 reading value that was significantly higher than that of the subtype reagents added to other wells.
  • the antibody subtype of antibodies 2DG5 and 1IB10 was IgG1.
  • the steps are as follows: Dilute the MCR-1 protein and MCR-2 protein to 0.2ug/mL, 100ul/well, and set up no coating control at the same time, and coat overnight at 4°C. Dried, washed 3 times with PBST; 5% skimmed milk powder, 200ul/well, blocked at 37°C for 2h; dried, washed 3 times with PBST, and added antibody diluted from 1:1000 times (concentration: 1mg/ml ), a total of 12 gradients, and at the same time set up an uncoated control 100ul/well, 37°C, 1h.
  • SDS-PAGE method was used to identify the molecular weight and purity of the antibody; gel preparation, separating gel was 12%, stacking gel was 5%; sample preparation, 20ul sample + 20ul buffer, mixed, boiled for 3min; each well was loaded with 20ul, and set up at the same time Protein pre-stained Marker control; 80 volts for 30 minutes, 120 volts for 2 hours; after electrophoresis, put in Coomassie brilliant blue solution for staining; decolorize, boil deionized water for 5 minutes each time, a total of 3 times; molecular weight of heavy chain of IgG antibody Generally, it is 50-75KDa, and the molecular weight of IgG antibody light chain is about 25KDa.
  • the purified monoclonal antibody is identified by SDS-PAGE; Clear bands.
  • the Ig variable region gene was cloned by RT-PCR, total RNA was extracted, and single-stranded cDNA was synthesized; the total RNA of 1IB10 and 2DG5 hybridoma cell lines was extracted by Trizol method (kit was purchased from Invitrogen), and M-MLV reverse transcriptase ( purchased from Invitrogen) to reverse the total RNA into a cDNA library.
  • cDNA 2 ⁇ l; Upstream primer (10 ⁇ M): 2 ⁇ l; Downstream primer (10 ⁇ M): 2 ⁇ l; dNTP mixture: 2 ⁇ l; pfu DNA polymerase (5U/ ⁇ l): 1 ⁇ l; 10 ⁇ pfu BufferII: 5 ⁇ l ; 50 ⁇ l.
  • Reaction conditions 95°C pre-denaturation for 5 minutes; repeat the following cycle 35 times: 95°C for 30s, 58°C for 30s, 72°C for 1min; finally, 72°C for 10min.
  • VL and VH fragments were separated and recovered by agarose gel electrophoresis.
  • the recovered VL and VH fragments were respectively connected to the pMD19-T(sKPCle) vector (Takara Company), and the connection system was as follows:
  • the ligation product was transformed into E.coli DH5 ⁇ competent bacteria. After culturing overnight at 37°C, a single colony was picked and shaken at 37°C for 2 hours for PCR identification of the bacteria solution.
  • the cDNA corresponding to the antibody was used as a positive control.
  • the PCR-positive clones of the selected bacteria were expanded and cultured, and the plasmids of the positive clones were extracted with a plasmid extraction kit (Takara Company) and sent for sequencing.
  • a plasmid extraction kit (Takara Company)
  • For each chain of each antibody send at least 5 clone samples for inspection, and at least three samples have the same sequencing results.
  • the heavy chain and light chain variable region sequences of antibodies 1IB10 and 2DG5 were successfully cloned, which were consistent with the sequence characteristics of typical antibody variable regions after comparison.
  • Embodiment 4 Preparation of MCR-1/MCR-2 protein detection card by colloidal gold method
  • Step 1 Add 0.1M K 2 CO 3 solution to the colloidal gold solution while stirring, adjust the pH value and add anti-MCR-1 monoclonal antibody 1IB10, after stirring, add 10% bovine serum albumin solution, 2% PEG20000, stir at low speed Centrifuge to take the supernatant, and then take the precipitate after high-speed centrifugation, and use colloidal gold resuspension to make up volume to form gold-labeled antibody;
  • Step 2 Spray the gold-labeled antibody on the glass cellulose membrane, and dry to make a gold-labeled pad;
  • Step 3 Add 1% sodium thimerosal solution to the mouse anti-MCR-1/MCR-2 protein monoclonal antibody 2DG5, mix well to form a detection line coating solution, and then add PBS and 1% thimerosal to the goat anti-mouse IgG Sodium solution, after mixing to form a quality control line coating solution, draw the quality control line coating solution and the detection line coating solution on the nitrocellulose membrane, and obtain the coating film after drying;
  • Step 4 Paste the coated film on the bottom plate, put the gold label pad and absorbent paper on the coated film, laminate and cut to obtain the colloidal gold method MCR-1/MCR-2 protein detection card.
  • the test results of the spotted test card were negative, and the spotting results of MCR-1 antigen, MCR-2 antigen, positive sample 1 containing MCR-1 protein, and positive sample 2 containing MCR-2 protein were all positive, which proves that the The colloidal gold method MCR-1/MCR-2 protein detection card can detect MCR-1 protein or MCR-2 protein and corresponding positive samples.

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Abstract

本发明提供鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，单克隆抗体及应用，通过小鼠杂交瘤单克隆抗体筛选及RT-PCR法克隆Ig可变区基因，获得稳定分泌鼠抗MCR-1/MCR-2蛋白抗体的杂交瘤细胞株及其可变区序列；通过系统性评价，鼠抗MCR-1/MCR-2蛋白抗体在各方面均有较佳表现，效价达到了1:1280000以上，适合作为免疫诊断试剂用于制备多粘菌素耐药菌株体外诊断试剂盒。

Description

一种鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，单克隆抗体及应用 技术领域
本发明属于抗体制备技术领域，尤其是涉及一种鼠抗MCR-1、MCR-2蛋白杂交瘤细胞株，单克隆抗体及应用。
背景技术
多黏菌素(polymyxin)是目前用于多重耐药和泛耐药革兰氏阴性细菌感染临床治疗的最后选择，近年来由于多粘菌素的临床使用增加，耐药菌株数量也逐年增加，虽然目前细菌的对多粘菌素耐药性并不强，却可能导致无药可医的地步，如何有效遏制耐药性的蔓延成为一个全球公关卫生问题。在多种药机制中，介导脂质A磷酸乙醇胺(PEtn)添加相关MCR基因被发现在多种质粒中，并可通过质粒在不同细菌中水平传播，甚至可以与其他耐药基因共同存在于同一质粒，表达后产生多种耐药机制。加之抗生素的不合理应用使得细菌耐药能力不断增强，给临床医师选用抗生素造成了极大的困扰。
实验室检测MCR介导的多黏菌素耐药主要是通过分子生物学法。研发具有自主知识产权的MCR蛋白快速诊断产品对于快速鉴定多黏菌素耐药菌株及其耐药机制，优化多黏菌素治疗方案，延长多黏菌素作为最后一线治疗药物的使用寿命具有重要意义。
发明内容
为解决上述技术问题，本发明提供一种鼠抗MCR-1、MCR-2蛋白杂交瘤细胞株，单克隆抗体及应用。
本发明采用的技术方案是：鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，命名为2DG5，保藏编号为CGMCCNo.23034；或者命名为1IB10，保藏编号为CGMCCNo.23032。
鼠抗MCR-1/MCR-2蛋白抗体，抗体2DG5，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；
SEQ ID NO:1 RASQDINNYLN(CDRL1)
SEQ ID NO:2 YTSRLHS(CDRL2)
SEQ ID NO:3 QQGKTIHPWT(CDRL3)
SEQ ID NO:4 GYSITSDYAWN(CDRH1)
SEQ ID NO:5 QISDSAVTTYNPSLKS(CDRH2)
SEQ ID NO:6 RNFYGYNFFDY(CDRH3)
或者，
抗体1IB10，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3；
SEQ ID NO:11 RSSQNIVCSYGNPCLE(CDRL1)
SEQ ID NO:12 KVSNRFS(CDRL2)
SEQ ID NO:13 FQSSHVPWT(CDRL3)
SEQ ID NO:14 GYTFTSYVMH(CDRH1)
SEQ ID NO:15 YFNPYNDGANYNEKFKG(CDRH2)
SEQ ID NO:16 GPYGNYAMDY(CDRH3)
优选地，抗体2DG5轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
SEQ ID NO:7
Figure PCTCN2022141244-appb-000001
SEQ ID NO:9
Figure PCTCN2022141244-appb-000002
或者，
抗体1IB10轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:17
Figure PCTCN2022141244-appb-000003
SEQ ID NO:19
Figure PCTCN2022141244-appb-000004
优选地，抗体2DG5由保藏编号为CGMCCNo.23034的鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株产生；
或者，
抗体1IB10由保藏编号为CGMCCNo.23032的鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株产生。
一种核酸分子，包含编码鼠抗MCR-1/MCR-2蛋白抗体的核苷酸。
优选地，所述核酸分子编码抗体2DG5的轻链可变区的核苷酸序列如SEQIDNO:8所示，所述核酸分子编码抗体2DG5的重链可变区的核苷酸序列如SEQIDNO:10所示；
SEQIDNO:8
Figure PCTCN2022141244-appb-000005
SEQIDNO:10
Figure PCTCN2022141244-appb-000006
Figure PCTCN2022141244-appb-000007
或者，
所述核酸分子编码抗体1IB10的轻链可变区的核苷酸序列如SEQIDNO:18所示，所述核酸分子编码抗体1IB10的重链可变区的核苷酸序列如SEQIDNO:20所示；
SEQIDNO:18
Figure PCTCN2022141244-appb-000008
SEQIDNO:20
Figure PCTCN2022141244-appb-000009
鼠抗MCR-1/MCR-2蛋白抗体在制备检测MCR-1或MCR-2蛋白试剂中的应用。
优选地，将鼠抗MCR-1/MCR-2蛋白抗体用于制备体外诊断试剂盒或微流体 芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
优选地，制备双抗体夹心法免疫胶体金试纸条，抗体2DG5为包被抗体，抗体1IB10为金标抗体；
或者，抗体1IB10为包被抗体，抗体2DG5为金标抗体。
本发明具有的优点和积极效果是：本发明提供两种鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，分别能够产生两种鼠抗MCR-1/MCR-2蛋白抗体，这两种抗体均能与MCR-1蛋白或MCR-2蛋白发生特异性反应；通过系统性评价，包括对抗体亚型及效价、试剂盒灵敏度、特异性和稳定性的评价，鼠抗MCR-1/MCR-2蛋白单克隆抗体在各方面均有较佳表现，效价达到了1:1280000以上，从而适合作为免疫诊断试剂用于制备检测多黏菌素耐药菌株体外诊断试剂盒。
附图说明
图1是鼠抗MCR-1/MCR-2蛋白蛋白电泳图；
图2是鼠抗MCR-1/MCR-2蛋白抗体蛋白电泳图；
图3胶体金法鼠抗MCR-1/MCR-2蛋白检测卡检测结果。
生物材料：2DG5，保藏日期为2021年9月24日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.23034；
生物材料：1IB10，保藏日期为2021年9月24日，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号是CGMCC No.23032。
具体实施方式
下面对本发明的实施例做出说明。
本发明涉及鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，生物材料命名为2DG5，属杂交瘤细胞，其保藏编号是CGMCC No.23034；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年9月24日，检测为存活。另有一株鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，生物材料命名为1IB10，属杂交瘤细胞，其保藏编号是CGMCC No.23032；保藏地为中国微生物菌种保藏管理委员会普通微生物中心，其保藏日期为2021年9月24日，检测为存活。
鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株生产得到的抗体2DG5，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID  NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；
SEQ ID NO:1 RASQDINNYLN(CDRL1)
SEQ ID NO:2 YTSRLHS(CDRL2)
SEQ ID NO:3 QQGKTIHPWT(CDRL3)
SEQ ID NO:4 GYSITSDYAWN(CDRH1)
SEQ ID NO:5 QISDSAVTTYNPSLKS(CDRH2)
SEQ ID NO:6 RNFYGYNFFDY(CDRH3)
抗体2DG5轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
SEQ ID NO:7
Figure PCTCN2022141244-appb-000010
SEQ ID NO:9
Figure PCTCN2022141244-appb-000011
编码抗体2DG5的轻链可变区的核苷酸序列如SEQIDNO:8所示，编码抗体2DG5的重链可变区的核苷酸序列如SEQIDNO:10所示；
SEQIDNO:8
Figure PCTCN2022141244-appb-000012
SEQIDNO:10
Figure PCTCN2022141244-appb-000013
鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株生产得到的抗体1IB10，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3；
SEQ ID NO:11 RSSQNIVCSYGNPCLE(CDRL1)
SEQ ID NO:12 KVSNRFS(CDRL2)
SEQ ID NO:13 FQSSHVPWT(CDRL3)
SEQ ID NO:14 GYTFTSYVMH(CDRH1)
SEQ ID NO:15 YFNPYNDGANYNEKFKG(CDRH2)
SEQ ID NO:16 GPYGNYAMDY(CDRH3)
抗体1IB10轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示；
SEQ ID NO:17
Figure PCTCN2022141244-appb-000014
SEQ ID NO:19
Figure PCTCN2022141244-appb-000015
编码抗体1IB10的轻链可变区的核苷酸序列如SEQIDNO:18所示，编码抗体1IB10的重链可变区的核苷酸序列如SEQIDNO:20所示；
SEQIDNO:18
Figure PCTCN2022141244-appb-000016
SEQIDNO:20
Figure PCTCN2022141244-appb-000017
鼠抗MCR-1/MCR-2蛋白单克隆抗体通过系统性评价，包括对抗体亚型及效价、试剂盒灵敏度、特异性和稳定性的评价；MCR-1蛋白和MCR--2蛋白较为相似，本发明制备得到的鼠抗MCR-1/MCR-2蛋白单克隆抗体既能够特异性识别MCR-1蛋白也能够特异性识别MCR-2蛋白，并在各方面均有较佳表现，从而适合作为免疫诊断试剂用于制备体外诊断试剂盒。可以制作成胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒，或者制作为微流体芯片；制作的试剂盒能够检测MCR-1、MCR-2蛋白。本方案所涉及的两种抗体尤其适合搭配形成双抗体夹心法免疫胶体金试纸条，其中抗体2DG5为包被抗体，抗体1IB10为金标抗体，制得的胶体金试纸条灵敏性更高，同时，也可将抗体2DG5做为金标抗体，抗体1IB10为包被抗体。
下面通过具体实施例对本发明做出进一步说明。其中，未具体说明操作步骤的实验方法，均按照相应商品说明书进行，实施例中所用到的仪器、试剂、耗材如无特殊说明，均可从商业公司购买得到。
实施例1：鼠抗MCR-1/MCR-2蛋白单克隆抗体的制备
1.1抗原制备
从NCBI中查找并下载mcr-1与mcr-2型基因序列，分别将重组质粒转化到大肠杆菌中进行诱导表达；纯化方法为镍柱亲和层析，菌体重悬于平衡缓冲液pH7.4中，超声至菌液澄清，高速离心，上清过膜后过镍柱，洗脱出目的蛋白，分子量和预计相符，SDS-PAGE如图1-2所示，用BCA法定量后分装。
1.2小鼠免疫
用纯化的MCR-1蛋白免疫6周龄左右的雌性Balb/c小鼠，进行抗体制备，按照免疫剂量分为2组，每组5只小鼠；按照抗原含量计算，第一组免疫剂量为25ug/只，第二组免疫剂量为50ug/只，首免，取适量MCR-1蛋白经生理盐水稀释至500ul，加入等量弗氏完全佐剂500ul乳化均匀，皮下多点注射免疫小鼠；两周后，取相同剂量进行二免，二免将佐剂换为弗氏不完全佐剂，并腹腔注射免疫小鼠，两周后再追加免疫一次，亦为腹腔注射免疫小鼠，7天后鼠尾采血，ELISA测定小鼠血清效价。具体步骤为：MCR-1蛋白0.2ug/ml，100ul/孔，4℃过夜包被ELISA板，甩干，PBST洗涤3次。5％脱脂乳粉，200ul/孔，37度封闭2h。小鼠鼠尾采血，3000转/min，离心后收集血清，从1:1000开始用PBS进行倍比稀释至1:512000，备用。甩干，PBST洗涤3次，1:1000倍起加入PBS稀释的一抗，100ul/孔，37度，1h。甩干，PBST洗涤3次,加PBS 1:10000倍稀释的羊抗鼠二抗，100ul/孔，37度，45min。甩干，PBST洗涤5次，加100ulTMB/孔，37℃，显色10min，终止，读值。
1.3细胞融合
融合前三天进行小鼠加强免疫，接种量同前次免疫，不加佐剂，腹腔注射。融合前一天准备饲养层细胞，取6-8周龄小鼠Balb/c 1只，取眼球放血后颈椎脱位致死，放于75％酒精中消毒5min，固定于盘上，在超净台中无菌剪开腹部皮肤。用无菌注射器吸取HAT选择培养液10ml注入小鼠腹腔，用酒精棉球轻揉腹 部，抽回培养基。加入40ml HAT培养液中，铺入到4块96孔细胞培养板中，100μL/孔，37℃，5％CO 2细胞培养箱中培养。融合前一周复苏骨髓瘤细胞(Sp2/0细胞)，用含10％胎牛血清的PRMI-1640培养基培养，37℃，5％CO 2培养箱中传代培养。将处于对数生长期的细胞收集至离心管中，细胞计数，把细胞稀释为10 7个/ml备用。取加强免疫3天的Balb/c小鼠，摘眼球放血制备阳性血清，脱颈椎处死，75％酒精消毒5min，在超净工作台无菌取出脾脏，在无菌平皿中冼涤数次，剥离结缔组织。将脾脏放在微孔铜网上，加入新鲜的RPMI-1640培养液，先用注射器吸取培养液由脾脏一段注入，吹下脾细胞，反复数次之后，用注射器的内塞轻轻将剩余脾脏研磨，直到无明显的红色组织块。将平皿中脾细胞悬液轻轻吹打后转移到50ml离心管中，1000r/min离心5min，收集脾细胞，计数后备用。将免疫鼠脾细胞与Sp2/0细胞按细胞数量10:1混合，加入50ml的离心管内，1000r/min离心5min，弃上清，在手心轻轻摩擦使两种细胞充分混匀，将离心管至于100mL蓝盖瓶内，蓝盖瓶内装有37℃热水，将预热好的1ml DMSO/PEG在1min内逐滴加入融合管内，先慢后快，边加边轻轻旋转离心管。然后立即加入无抗无血RPMI-1640培养液终止反应，第一分钟加1ml，第二分钟加2ml，第三分钟加3ml，第四分钟加4ml。37℃水浴5min，后800r/min离心5min，弃上清，将沉淀以HAT悬起，混匀到40ml含37℃预热的20％小牛血清的HAT选择培养液中，铺入已加有饲养细胞的96孔细胞板中，100μL/孔，将培养板放入37℃，5％CO 2培养箱培养。7d后将用新鲜的HAT培养基对细胞板半换液，10天后用HT培养基全换液。将96孔板中检测阳性的细胞采用有限稀释法进行亚克隆：首先按照上述方法制备饲养层细胞，取待克隆杂交瘤细胞进行细胞计数，用HT培养基将细胞稀释至5-8个细胞/ml，加入到已铺饲养细胞的96孔细胞板中100μL/孔，每株杂交瘤细胞克隆一块96孔细胞板，37℃、5％CO 2细胞培养箱中培养。约5天后数出细胞孔里的克隆数，标记，7天时并换新的培养基，待细胞铺满整个孔底的1/3～1/2时检测。经过2-3次克隆化，待96孔板所有细胞孔均为阳性时，即可进行扩大培养，定株，冻存。将检测阳性确定定株的杂交瘤细胞扩大培养并冻存。具体过程如下：将生长旺盛、状态良好的杂交瘤细胞用无抗无血DMEM轻轻从细胞瓶上吹下，1000r/min离心5min，弃去上清。加入冻存液(含40％RPMI-1640培养液、50％胎牛血清、10％DMSO)，将细胞 吹散后分装到细胞冻存管中。将冻存管放入冻存盒置于-70℃冰箱中，一天后将冻存管转移入液氮中，做好记录。
1.4腹水制备
取10-12周龄雌性Balb/c小鼠，腹腔注射无菌液体石蜡，0.5mL/只，7d后腹腔注射培养至对数期的杂交瘤细胞，5×10 6个细胞/只。每天注意观察，约7-10天，待小鼠腹部出现明显隆起后，用75％酒精棉球消毒下腹部皮肤，用16号针头刺入腹腔，收集腹水。待腹水再生积聚后，再次收集。将收集的腹水3000r/min离心10min，取中间澄清部分，用滤纸过滤后，分装，-70℃保存。
1.5抗体纯化
用Protein-G柱子进行腹水纯化，步骤如下：取腹水2ml，10000g离心，取澄清部分，加入2ml洗涤缓冲液，混匀，柱子用20％乙醇流尽后用8mL洗涤液平衡，样本过柱子，流速为8S/滴，反复上样3次，然后用15mL洗涤缓冲液进行洗涤淀，流速为8S/滴，洗涤完毕后用10mL的洗脱缓冲液进行洗脱，洗脱完毕会用1M Tris PH＝9调PH至7.4，然后用浓缩注进行浓缩，于50kd透析袋，PBS，4℃透析过夜。
实施例2：鼠抗MCR-1、MCR-2蛋白单克隆抗体的鉴定
2.1抗体亚类鉴定
按照SIGMA试剂盒说明书，以捕获ELISA的方法进行单抗的亚类鉴定，具体如下：将单抗亚类鉴定试剂1:1000稀释后，加入酶标孔中，100μL/孔，37℃孵育1h；PBST洗三次，拍干；将抗体1:1000倍稀释后加样，100μL/孔，37℃孵育1h；PBST洗三次，拍干；HRP酶标羊抗鼠IgG二抗以1:10000稀释后加样，100μL/孔，室温孵育30min；显色10～20min。以OD 450读值明显高于其他孔所加亚类试剂为单抗所属亚类类型。抗体2DG5、1IB10的抗体亚型为IgG1。
2.2抗体效价测定
采用间接ELISA法进行纯化后抗体效价测定，步骤如下：分别将MCR-1蛋白和MCR-2蛋白稀释至0.2ug/mL，100ul/孔，同时设立不包被对照，4℃过夜包被，甩干，PBST洗涤3次；5％脱脂乳粉，200ul/孔，37度封闭2h；甩干，PBST洗涤3次，加入从1:1000倍开始进行倍比稀释的抗体(浓度为1mg/ml)，共计12个梯度， 同时设立不包被对照100ul/孔，37℃，1h。甩干，PBST洗涤3次，加PBS 1:10000倍稀释的羊抗鼠二抗，100ul/孔，37℃，45min。甩干，PBST洗涤5次,加100ulTMB/孔，37℃，显色10min，终止，读值。纯化后抗体稀释至1mg/ml，针对MCR-1蛋白和MCR-2蛋白分别进行效价检测，效价均达到了1:1280000以上。
2.3抗体纯度及分子量鉴定
采用SDS-PAGE法进行抗体分子量及纯度鉴定；制胶，分离胶为12％，浓缩胶为5％；制样，20ul样品+20ul buffer，混匀，煮沸3min；每孔上样20ul，同时设立蛋白预染Marker对照；80伏30min，120伏2h；电泳完毕后，放入考马斯亮蓝溶液进行染色；脱色，去离子水煮沸脱色，每次5min，共计3次；lgG抗体重链的分子质量一般为50-75KDa，lgG抗体轻链的分子量约为25KDa，通过SDS-PAGE对纯化得到的单克隆抗体进行鉴定；如图2所示，抗体2DG5和抗体1IB10在50-75KDa和约25KDa处各有清晰的条带。
实施例3：鼠抗MCR-1、MCR-2蛋白单克隆抗体的基因验证
RT-PCR法克隆Ig可变区基因，提取总RNA，合成单链cDNA；用Trizol法(试剂盒购自Invitrogen)提取1IB10,2DG5杂交瘤细胞株的总RNA，用M-MLV逆转录酶(购自Invitrogen)将总RNA逆转为cDNA文库。
重链骨架区上游引物
P1：5’SAGGTGMAGCTKCASSARTCWGG3’
重链可变区下游引物
P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’
轻链前导肽上游引物
P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’
轻链可变区下游引物
P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’
配制PCR反应体系(50μl)如下：
cDNA:2μl；上游引物(10μM):2μl；下游引物(10μM):2μl；dNTP mixture:2μl；pfu DNA聚合酶(5U/μl):1μl；10×pfu BufferⅡ:5μl；ddH 2O:补足至50μl。
反应条件：95℃预变性5min；重复如下循环35次：95℃30s，58℃30s，72℃1min； 最后，72℃延伸10min。
琼脂糖凝胶电泳分离并回收VL、VH片段。将回收后的VL、VH片段分别与pMD19-T(sKPCle)载体(Takara公司)进行连接，连接体系如下：
VL PCR产物/VH PCR产物各70ng，pMD19-T(sKPCle)载体1μl，Solution I连接反应液5μl；ddH2O补足至10μl，4℃连接过夜。
连接产物转化入E.coli DH5α感受态细菌中，37℃过夜培养后，挑取单个菌落，37℃震摇2小时后进行菌液PCR鉴定，以对应抗体的cDNA为阳性对照。配制反应体系(25μl)如下：
菌液：1μl，上游引物(10μM)：1μl；下游引物(10μM)：1μl；dNTP Mixture(各2.5Mm)2μl；Taq DNA聚合酶(5U/μl)：0.5μl；10×Taq Buffer(Mg 2+plus)：2.5μl；补水至25μl。反应条件同前。
选取菌PCR阳性的克隆扩大培养，用质粒提取试剂盒(Takara公司)提取阳性克隆质粒，送检测序。每个抗体的每条链至少送检5个克隆样品，至少三个样品测序结果相同为止。成功克隆得到抗体1IB10、2DG5的重链、轻链可变区序列，经比对符合典型抗体可变区序列特征。
实施例4：胶体金法制备MCR-1/MCR-2蛋白检测卡
制备双抗体夹心法免疫胶体金试纸条，制备方法为：
步骤一向胶体金溶液中边搅拌边加入0.1M K 2CO 3溶液，调节pH值后加入抗MCR-1单克隆抗体1IB10，搅拌后加入10％的牛血清白蛋白溶液，2％PEG20000，搅拌后低速离心取上清，再高速离心后取沉淀，用胶体金重悬液定容形成金标抗体；
步骤二将金标抗体喷于玻璃纤维素膜，烘干制成金标垫；
步骤三向鼠抗MCR-1/MCR-2蛋白单克隆抗体2DG5中加入1％的硫柳汞钠溶液，混匀后形成检测线包被液，再向羊抗鼠IgG中加入PBS和1％的硫柳汞钠溶液，混匀后形成质控线包被液，将质控线包被液和检测线包被液划在硝酸纤维素膜上，烘干后获得包被膜；
步骤四将包被膜贴在底板上，将金标垫和吸水纸搭上包被膜，层压后切割获得胶体金法MCR-1/MCR-2蛋白检测卡。
取通过上述方法制备得到的胶体金法MCR-1/MCR-2蛋白检测卡，分别取空白样、MCR-1抗原、MCR-2抗原、含有MCR-1蛋白阳性样本1和含有MCR-2蛋白阳性样本2点样于检测卡。结果如图3所示，从左到右依次为空白样、MCR-1抗原、MCR-2抗原、含有MCR-1蛋白阳性样本1和含有MCR-2蛋白阳性样本2，能够看出，空白样点样的检测卡检测结果呈阴性，MCR-1抗原、MCR-2抗原、含有MCR-1蛋白阳性样本1和含有MCR-2蛋白阳性样本2点样结果均为阳性，证明通过本方案制备得到的胶体金法MCR-1/MCR-2蛋白检测卡能够检测MCR-1蛋白或MCR-2蛋白及相应阳性样本。
以上对本发明的实施例进行了详细说明，但所述内容仅为本发明的较佳实施例，不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等，均应仍归属于本发明的专利涵盖范围之内。

Claims (9)

  1. 鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株，其特征在于：命名为2DG5，保藏编号为CGMCCNo.23034；或者命名为1IB10，保藏编号为CGMCCNo.23032。
  2. 鼠抗MCR-1/MCR-2蛋白抗体，其特征在于：抗体2DG5，包括轻链可变区和重链可变区，轻链可变区包括如SEQ ID NO:1所示的CDRL1、SEQ ID NO:2所示的CDRL2和SEQ ID NO:3所示的CDRL3，重链可变区包括如SEQ ID NO:4所示的CDRH1、SEQ ID NO:5所示的CDRH2和SEQ ID NO:6所示的CDRH3；或者，
    抗体1IB10，轻链可变区包括如SEQ ID NO:11所示的CDRL1、SEQ ID NO:12所示的CDRL2和SEQ ID NO:13所示的CDRL3，重链可变区包括如SEQ ID NO:14所示的CDRH1、SEQ ID NO:15所示的CDRH2和SEQ ID NO:16所示的CDRH3。
  3. 根据权利要求2所述的鼠抗MCR-1/MCR-2蛋白抗体，其特征在于：抗体2DG5轻链可变区氨基酸序列为SEQ ID NO:7所示，重链可变区氨基酸序列为SEQ ID NO:9所示；
    或者，
    抗体1IB10轻链可变区氨基酸序列为SEQ ID NO:17所示，重链可变区氨基酸序列为SEQ ID NO:19所示。
  4. 根据权利要求2或3所述的鼠抗MCR-1/MCR-2蛋白抗体，其特征在于：抗体2DG5由保藏编号为CGMCCNo.23034的鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株产生；
    或者，
    抗体1IB10由保藏编号为CGMCCNo.23032的鼠抗MCR-1/MCR-2蛋白杂交瘤细胞株产生。
  5. 一种核酸分子，其特征在于：包含编码权利要求2或3所述的鼠抗MCR-1/MCR-2蛋白抗体的核苷酸。
  6. 根据权利要求5所述的核酸分子，其特征在于：所述核酸分子编码抗体2DG5的轻链可变区的核苷酸序列如SEQIDNO:8所示，所述核酸分子编码抗体2DG5的重链可变区的核苷酸序列如SEQIDNO:10所示；
    或者，
    所述核酸分子编码抗体1IB10的轻链可变区的核苷酸序列如SEQIDNO:18所示，所述核酸分子编码抗体1IB10的重链可变区的核苷酸序列如SEQIDNO:20所示。
  7. 权利要求2-4中任一所述的鼠抗MCR-1/MCR-2蛋白抗体在制备检测MCR-1或MCR-2蛋白试剂中的应用。
  8. 根据权利要求7所述的鼠抗MCR-1/MCR-2蛋白抗体在制备检测MCR-1或MCR-2蛋白试剂中的应用，其特征在于：将鼠抗MCR-1/MCR-2蛋白抗体用于制备体外诊断试剂盒或微流体芯片，所述体外诊断试剂盒为胶体金免疫试剂盒、化学发光试剂盒、放射免疫试剂盒、酶联免疫试剂盒或荧光免疫试剂盒。
  9. 根据权利要求7所述的鼠抗MCR-1/MCR-2蛋白抗体在制备检测MCR-1或MCR-2蛋白试剂中的应用，其特征在于：制备双抗体夹心法免疫胶体金试纸条，抗体2DG5为包被抗体，抗体1IB10为金标抗体；
    或者，抗体1IB10为包被抗体，抗体2DG5为金标抗体。
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