CN114317455B

CN114317455B - Mouse anti-MCR-1 protein hybridoma cell strain, monoclonal antibody and application

Info

Publication number: CN114317455B
Application number: CN202210194929.7A
Authority: CN
Inventors: 李曦; 李可可; 陈晓玲; 王亚苗; 孔迪; 王思怡; 王芮; 王兴; 臧丹戎
Original assignee: Beijing Gold Mountainriver Tech Development Co ltd; Tianjin Xinuo Biological Pharmaceutical Co ltd; Tianjin Era Biology Technology Co ltd
Current assignee: Beijing Gold Mountainriver Tech Development Co ltd; Tianjin Era Biology Technology Co ltd; Tianjin Xinuo Biological Pharmaceutical Co ltd; Zhejiang Provincial Peoples Hospital
Priority date: 2022-03-02
Filing date: 2022-03-02
Publication date: 2022-05-31
Anticipated expiration: 2042-03-02
Also published as: CN114317455A

Abstract

The invention provides a mouse anti-MCR-1 protein hybridoma cell strain, a monoclonal antibody and application, wherein Ig variable region genes are cloned by mouse hybridoma monoclonal antibody screening and an RT-PCR method to obtain a hybridoma cell strain capable of stably secreting a mouse anti-MCR-1 protein antibody and a variable region sequence thereof; through systematic evaluation, the mouse anti-MCR-1 protein antibody has better performance in all aspects, the titer reaches more than 1:1280000, and the mouse anti-MCR-1 protein antibody is suitable to be used as an immunodiagnostic reagent for preparing a polymyxin drug-resistant strain in-vitro diagnosis kit.

Description

Mouse anti-MCR-1 protein hybridoma cell strain, monoclonal antibody and application

Technical Field

The invention belongs to the technical field of antibody preparation, and particularly relates to a mouse anti-MCR-1 protein hybridoma cell strain, a monoclonal antibody and application.

Background

Polymyxin is a cationic polypeptide antibiotic, acts on lipopolysaccharide of gram-negative bacteria cell wall, destroys bacterial cell structure, and is the most effective antibiotic for treating carbapenem drug-resistant gram-negative bacteria. In recent years, the number of drug-resistant strains has been increasing year by year due to the increasing clinical use of polymyxins. Among the various drug mechanisms, MCR-1 belongs to the phosphoethanolamine transferase family, and MCR-1 protein modifies lipid a, resulting in a decrease in the affinity of polymyxin for bacterial lipopolysaccharide, thereby mediating bacterial resistance to polymyxin. Meanwhile, the mcr-1 gene is horizontally transmitted in different bacteria through plasmids, and can even coexist with other drug-resistant genes in the same plasmid to generate a plurality of drug-resistant mechanisms after expression. Therefore, it is very important to establish a rapid and accurate detection method of mcr-1 drug-resistant strains.

At present, detection of mcr-1 drug-resistant strains is mainly achieved through molecular biology means, but the detection is inconvenient to popularize due to high requirements on detection equipment and environment. The immunological analysis method has the advantages of high sensitivity, strong specificity, short period, simple operation, low cost and the like, wherein the enzyme-linked immunosorbent assay is most commonly used. Aiming at the situation, MCR-1 protein is extracted as an antigen, after the high-purity antigen is obtained, a good immune reaction is stimulated in a mouse body, a hybridoma technology is further adopted to screen a monoclonal antibody with high affinity and specificity, a double-antibody sandwich ELISA method is established on the basis, and the quick detection of the MCR-1 drug-resistant strain is realized.

Disclosure of Invention

In order to solve the technical problems, the invention provides a mouse anti-MCR-1 protein hybridoma cell strain, a monoclonal antibody and application.

The technical scheme adopted by the invention is as follows: the mouse MCR-1 protein hybridoma cell strain is named as 1HD4, and the preservation number is CGMCC No. 23031; or 1JA9 with the preservation number of CGMCC No. 23033.

A murine anti-MCR-1 protein antibody, antibody 1HD4, comprising a light chain variable region comprising CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2 and CDRL3 as shown in SEQ ID NO:3 and a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5 and CDRH3 as shown in SEQ ID NO: 6;

SEQ ID NO:1 RASQDISNYLN （CDRL1）

SEQ ID NO:2 YTSRLHS（CDRL2）

SEQ ID NO:3 QQGNTFPYT（CDRL3）

SEQ ID NO:4 GYIFTCCKMY（CDRH1）

SEQ ID NO:5 YFDPYNGDTSSNQKFKG（CDRH2）

SEQ ID NO:6 WLQNYYAMDY（CDRH3）

or,

antibody 1JA9, the light chain variable region comprising CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12 and CDRL3 as shown in SEQ ID NO:13, the heavy chain variable region comprising CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15 and CDRH3 as shown in SEQ ID NO: 16;

SEQ ID NO:11 RSSQNIVHSYGNPSLE（CDRL1）

SEQ ID NO:12 KVSNRFS（CDRL2）

SEQ ID NO:13 FQASHVPWT（CDRL3）

SEQ ID NO:14 GYTFTSYIMH（CDRH1）

SEQ ID NO:15 YFNPYTDGSKYNEMFNG（CDRH2）

SEQ ID NO:16 GPYGNYAMDY（CDRH3）

preferably, the amino acid sequence of the light chain variable region of the antibody 1HD4 is shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9;

SEQ ID NO:7

DVQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGADYSLTISNLEEEDIATYFCQQGNTFPYTFGGGTKLEIK

SEQ ID NO:9

VKLQESGPELVKPGASVKVSCKASGYIFTCCKMYWVKQSHGKSLEWIGYFDPYNGDTSSNQKFKGKVTLTVDKSSSTAYMFLNSLTSEDSAVYYCASWLQNYYAMDYWGQGTSVTVSS

the amino acid sequence of the light chain variable region of the antibody 1JA9 is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19;

SEQ ID NO:17

EILLTQTPLSLPVSLGDQASISCRSSQNIVHSYGNPSLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHVPWTFGGGTKLEIKRAD

SEQ ID NO:19

VKLQESGPELVKPGASVKMSCRTSGYTFTSYIMHWVKQKPGQGLEWIGYFNPYTDGSKYNEMFNGKATLSSDKSSGTAYMEFSSLTSEDSAVYYCARGPYGNYAMDYWGQGTSVTVSAAKT

preferably, the antibody 1HD4 is produced by a mouse anti-MCR-1 protein hybridoma cell line with the preservation number of CGMCC No. 23031;

the antibody 1JA9 is produced by a mouse anti-MCR-1 protein hybridoma cell strain with the preservation number of CGMCC No. 23033.

A nucleic acid molecule comprising nucleotides encoding a murine anti-MCR-1 protein antibody.

Preferably, the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of antibody 1HD4 is shown in SEQ ID NO:8, and the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of antibody 1HD4 is shown in SEQ ID NO: 10;

SEQ ID NO:8

GATGTCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAATTATTTAAATTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAGCAGATTATTCTCTCACCATCAGTAACCTGGAGGAGGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACATTTCCGTACACATTCGGAGGGGGGACCAAGCTGGAAATCAAA

SEQ ID NO:10

GTGAAGCTGCAGGAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGTTATATATTCACTTGCTGCAAAATGTACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATTTTGATCCTTACAATGGTGATACTAGTTCCAACCAGAAGTTCAAGGGCAAGGTCACATTGACTGTTGACAAGTCTTCCAGTACAGCCTACATGTTTTTGAACAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTGGCTACAAAATTACTATGCTATGGACTACTGGGGTCAAGGCACCTCAGTCACCGTCTCCTCA

the nucleotide sequence of the light chain variable region of the nucleic acid molecule coding antibody 1JA9 is shown as SEQ ID NO:18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule coding antibody 1JA9 is shown as SEQ ID NO: 20;

SEQ ID NO:18

GAGATCTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTTATGGAAACCCCTCTTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGCTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT

SEQ ID NO:20

GTGAAGCTTCAGGAATCTGGACCTGAGCTGGTTAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAGGACTTCTGGCTACACATTCACTAGCTATATTATGCACTGGGTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACACTGATGGTTCTAAGTACAATGAGATGTTCAACGGCAAGGCCACACTGTCCTCAGACAAATCCTCCGGCACAGCCTATATGGAGTTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCGAGAGGCCCCTATGGTAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGCAGCCAAAACG

application of a mouse anti-MCR-1 protein antibody in preparing a reagent for detecting MCR-1 protein.

Preferably, the mouse anti-MCR-1 protein antibody is used for preparing an in vitro diagnostic kit or a microfluidic chip, and the in vitro diagnostic kit is a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunosorbent assay kit or a fluorescence immunoassay kit.

Preferably, an enzyme-linked immunoassay kit is prepared, wherein the antibody 1HD4 is a coating antibody, and the antibody 1JA9 is an HRP-labeled antibody;

alternatively, antibody 1JA9 was the coating antibody and antibody 1HD4 was the HRP-labeled antibody.

The invention has the advantages and positive effects that: the invention provides two mouse anti-MCR-1 protein hybridoma cell strains which can respectively generate two mouse anti-MCR-1 protein antibodies; through systematic evaluation, including evaluation on antibody subtype and titer, kit sensitivity, specificity and stability, the mouse anti-MCR-1 protein monoclonal antibody has better performance in all aspects, and the titer reaches more than 1:1280000, so that the mouse anti-MCR-1 protein monoclonal antibody is suitable to be used as an immunodiagnostic reagent for preparing a polymyxin drug-resistant strain in-vitro diagnosis kit.

Drawings

FIG. 1 is an electrophoretogram of a murine anti-MCR-1 protein;

FIG. 2 is a protein electrophoretogram of a murine anti-MCR-1 protein antibody;

biological material: 1HD4, the preservation date is 2021, 9 and 24 months, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 23031;

biological material: 1JA9, the preservation date is 2021, 9 months and 24 days, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 23033.

Detailed Description

The following description is made of embodiments of the present invention.

The invention relates to a mouse MCR-1 protein-resistant hybridoma cell strain, which is a biological material named as 1HD4 and belongs to hybridoma cells, and the preservation number of the cell strain is CGMCC number 23031; the preservation place is China general microbiological culture Collection center, the preservation date is 2021, 9 months and 24 days, and the preservation place is detected to be alive. Another mouse MCR-1 protein hybridoma cell strain, wherein the biological material is named as 1JA9, belongs to hybridoma cells, and the preservation number is CGMCC No. 23033; the preservation place is China general microbiological culture Collection center, the preservation date is 2021, 9 months and 24 days, and the preservation place is detected to be alive.

The antibody 1HD4 produced by the mouse MCR-1 protein hybridoma cell strain comprises a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDRL1 shown in SEQ ID NO:1, CDRL2 shown in SEQ ID NO:2 and CDRL3 shown in SEQ ID NO:3, and the heavy chain variable region comprises CDRH1 shown in SEQ ID NO:4, CDRH2 shown in SEQ ID NO:5 and CDRH3 shown in SEQ ID NO: 6;

SEQ ID NO:1 RASQDISNYLN （CDRL1）

SEQ ID NO:2 YTSRLHS（CDRL2）

SEQ ID NO:3 QQGNTFPYT（CDRL3）

SEQ ID NO:4 GYIFTCCKMY（CDRH1）

SEQ ID NO:5 YFDPYNGDTSSNQKFKG（CDRH2）

SEQ ID NO:6 WLQNYYAMDY（CDRH3）

the amino acid sequence of the light chain variable region of the antibody 1HD4 is shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9;

SEQ ID NO:7

SEQ ID NO:9

the nucleotide sequence of the light chain variable region of the coded antibody 1HD4 is shown as SEQ ID NO. 8, and the nucleotide sequence of the heavy chain variable region of the coded antibody 1HD4 is shown as SEQ ID NO. 10;

SEQ ID NO:8

SEQ ID NO:10

antibody 1JA9 produced by mouse anti-MCR-1 protein hybridoma cell strain, light chain variable region comprises CDRL1 shown in SEQ ID NO:11, CDRL2 shown in SEQ ID NO:12 and CDRL3 shown in SEQ ID NO:13, heavy chain variable region comprises CDRH1 shown in SEQ ID NO:14, CDRH2 shown in SEQ ID NO:15 and CDRH3 shown in SEQ ID NO: 16;

SEQ ID NO:11 RSSQNIVHSYGNPSLE（CDRL1）

SEQ ID NO:12 KVSNRFS（CDRL2）

SEQ ID NO:13 FQASHVPWT（CDRL3）

SEQ ID NO:14 GYTFTSYIMH（CDRH1）

SEQ ID NO:15 YFNPYTDGSKYNEMFNG（CDRH2）

SEQ ID NO:16 GPYGNYAMDY（CDRH3）

SEQ ID NO:17

SEQ ID NO:19

the nucleotide sequence of the light chain variable region of the coded antibody 1JA9 is shown as SEQ ID NO. 18, and the nucleotide sequence of the heavy chain variable region of the coded antibody 1JA9 is shown as SEQ ID NO. 20;

SEQ ID NO:18

SEQ ID NO:20

the mouse anti-MCR-1 protein monoclonal antibody has better performance in all aspects through systematic evaluation, including evaluation on antibody subtype and titer, kit sensitivity, specificity and stability, so that the mouse anti-MCR-1 protein monoclonal antibody is suitable for being used as an immunodiagnostic reagent for preparing an in vitro diagnosis kit. Can be made into a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit, or can be made into a microfluid chip; the prepared kit can detect MCR-1 protein. The two antibodies related to the scheme are particularly suitable for being matched to form an enzyme-linked immunosorbent assay reagent, wherein the antibody 1HD4 is a coating antibody, the antibody 1JA9 is an HRP-labeled antibody, the sensitivity of the prepared enzyme-linked immunosorbent assay reagent is higher, meanwhile, the antibody 1HD4 can also be used as the HRP-labeled antibody, and the antibody 1JA9 is a coating antibody.

The invention is further illustrated by the following specific examples. The experimental methods without specific description of the operation steps are all performed according to corresponding commercial specifications, and instruments, reagents and consumables used in the examples can be purchased from commercial companies if no special description is provided.

Example 1: preparation of mouse anti-MCR-1 protein monoclonal antibody

1.1 antigen preparation

Searching and downloading an mcr-1 type gene sequence from NCBI, and transforming the recombinant plasmid into escherichia coli for induced expression; the purification method comprises the steps of nickel column affinity chromatography, bacterial liquid clarification by ultrasonic treatment of the bacteria, high-speed centrifugation, membrane passing of the supernatant, nickel column passing, elution of target protein, the molecular weight of the target protein being consistent with that of the expected 40.6KDa, and quantitative BCA (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) method shown in figure 1 of SDS-PAGE and subpackaging.

1.2 immunization of mice

Immunizing female Balb/c mice of about 6 weeks old with the purified MCR-1 protein, preparing antibodies, and dividing the antibodies into 2 groups according to the immune dose, wherein each group comprises 3 mice; according to the calculation of antigen content, the first group of immune dose is 25 ug/mouse, the second group of immune dose is 50 ug/mouse, first immunization, taking a proper amount of MCR-1 protein to be diluted to 500ul by normal saline, adding 500ul of equivalent Freund's complete adjuvant to be emulsified evenly, and injecting immune mice subcutaneously at multiple points; two weeks later, the same dose was taken for secondary immunization, the adjuvant was changed to Freund's incomplete adjuvant, and the immunized mice were injected intraperitoneally once again two weeks later, and blood was collected from the rat tail 7 days later, and the serum titer of the mice was measured by ELISA.

The method comprises the following specific steps: MCR-1 protein 0.2ug/ml, 100 ul/well, 4 degrees overnight coated ELISA plate, spin-dry, PBST washing 3 times. 5% of skim milk powder, 250 ul/hole, and sealing for 2 h. Blood is collected from mouse tails at 3000RPM, serum is collected after centrifugation, and diluted from 1:1000 with PBS to 1:5120000 for later use. Spin-dry, wash 3 times with PBST, add PBS diluted primary antibody, 100 ul/well, 37 degrees C, 1 h. PBST was washed 3 times, goat anti-mouse secondary antibody was added at 100 ul/well, 37 degrees, 40 min. PBST was washed 4 times, 100ul TMB/well was added, developed at 37 ℃ for 20min, stopped, and read.

1.3 cell fusion

Three days before fusion, mice are subjected to boosting immunization, the inoculation amount is the same as that of the previous immunization, and adjuvant is not added, and intraperitoneal injection is carried out. Preparing feeder layer cells one day before fusion, collecting Balb/c 1 mouse 6-8 weeks old, collecting eyeball for exsanguination, dislocating cervical vertebra, and killing, and placing at 75%Sterilized in alcohol, and aseptically cut open the abdominal skin in a clean bench. The mouse abdominal cavity was injected with 10ml of the HAT selection medium, and the abdomen was gently massaged with an alcohol cotton ball, and the medium was withdrawn. Adding into HAT culture solution 40ml, spreading into 96-well 4-well cell culture plates at a concentration of 100 μ L/well and 37 deg.C with 5% CO₂Culturing in a cell culture box. One week before fusion, myeloma cells (Sp 2/0 cells) were recovered and cultured in PRMI-1640 medium containing 10% fetal bovine serum at 37 ℃ under 5% CO₂Subculturing in an incubator. Collecting cells in logarithmic growth phase into a centrifuge tube, counting the cells, diluting the cells to 10⁷One/ml is ready for use. Collecting Balb/c mice with 3 days of enhanced immunity, picking eyeball, bleeding to prepare positive serum, removing cervical vertebra, killing, sterilizing with 75% alcohol for 5min, aseptically taking out spleen from ultra-clean bench, washing in aseptic petri dish for several times, and stripping connective tissue. Placing spleen on microporous copper net, adding fresh RPMI-1640 culture solution, sucking the culture solution with syringe, injecting into spleen section, blowing down splenocytes, repeating for several times, and lightly grinding the rest spleen with the inner plug of syringe until there is no obvious red tissue block. And gently blowing and beating the splenocyte suspension in the plate, transferring the splenocyte suspension into a 50ml centrifuge tube, centrifuging for 5min at 1000r/min, collecting splenocytes, and counting for later use. Mixing splenocytes of the immunized mice with Sp2/0 cells according to the cell number of 10:1, adding the mixture into a 50mL centrifuge tube, centrifuging the mixture for 5min at 1000r/min, discarding the supernatant, gently rubbing the mixture at the palm to fully mix the two cells, placing the centrifuge tube into a 100mL blue-covered bottle, filling hot water at 37 ℃ into the blue-covered bottle, dropwise adding preheated 1mL DMSO/PEG into the fusion tube within 1min, and slowly and quickly rotating the centrifuge tube while adding. Then, the reaction was terminated by immediately adding the non-resistant and bloodless RPMI-1640 culture medium, 1ml for the first minute, 2ml for the second minute, 3ml for the third minute, and 4ml for the fourth minute. Water bath at 37 deg.C for 5min, centrifuging at 800r/min for 5min, discarding supernatant, suspending the precipitate with HAT, mixing well into 40ml HAT selective culture solution containing 20% calf serum preheated at 37 deg.C, spreading into 96-well cell plate with feeder cells, placing the cell plate at 37 deg.C and 5% CO at 100 μ L/well, and culturing at 37 deg.C₂Culturing in an incubator. After 7d, the cell plates will be half-exchanged with fresh HAT medium and 10 days later with the HT medium. Checking the 96-well platePositive cells were subcloned by limiting dilution: firstly, preparing feeder layer cells according to the method, taking hybridoma cells to be cloned for cell counting, diluting the cells to 5-8 cells/ml by using HT culture medium, adding the cells into a 96-well cell plate with the laid feeder cells at 100 mu L/well, cloning one 96-well cell plate for each hybridoma cell, carrying out cell cloning at 37 ℃ by 5% CO₂Culturing in a cell culture box. After about 5 days, counting the number of clones in the cell wells, marking, changing the culture medium after 7 days, and detecting when the cells are fully paved at 1/3-1/2 of the whole well bottom. After 2-3 times of cloning, when all cell wells of the 96-well plate are positive, performing expanded culture, fixing the strains and freezing and storing. And (4) carrying out expanded culture on the hybridoma cells with positive detection and definite strains and freezing and storing. The specific process is as follows: the hybridoma cells which grow vigorously and are in good state are gently blown down from the cell bottle by using the anti-free blood-free DMEM, and are centrifuged at 1000r/min for 5min, and the supernatant is discarded. Adding freezing medium (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, and 10% DMSO), blowing out cells, and packaging into cell freezing tube. And (4) placing the freezing tube into a freezing box, placing the freezing box in a refrigerator at the temperature of-70 ℃, transferring the freezing tube into liquid nitrogen after one day, and recording.

1.4 preparation of ascites

Taking 10-12 week old female Balb/c mice, injecting sterile liquid paraffin into abdominal cavity, culturing to hybridoma cells of logarithmic phase in an injection manner, and culturing for 7 days, wherein each mouse is 0.5mL, and the number of hybridoma cells is 5 multiplied by 10⁶One cell/one. Observing every day, taking care about 7-10 days, after the abdomen of the mouse has obviously raised, disinfecting the skin of the abdomen with a 75% alcohol cotton ball, puncturing the abdominal cavity with a needle, and collecting ascites. And collecting the ascites again after the ascites is regenerated and accumulated. Centrifuging the collected ascites at 3000 r/min for 10min, collecting the intermediate clear part, filtering with filter paper, and packaging at-70 deg.C.

1.5 antibody purification

Ascites was purified using Protein-G column, the procedure was as follows: taking 2mL (n) of ascites, centrifuging 10000g, taking a clear part, adding 2mL (1: 1) of washing buffer solution, mixing uniformly, allowing the column to run out of 20% ethanol, then balancing with 8mL of washing solution, allowing the sample to pass through the column at the flow rate of 8S/drop, repeatedly loading for 3 times, then washing and precipitating with 15mL of washing buffer solution at the flow rate of 8S/drop, eluting with 10mL of elution buffer solution after washing, adjusting the pH to 7.4 with 1M Tris pH =9 after the elution is finished, then concentrating with a concentration column, dialyzing overnight at 4 ℃ in a 50kd dialysis bag and PBS.

Example 2: identification of mouse anti-MCR-1 protein monoclonal antibody

2.1 antibody subclass identification

Subclass identification of the monoclonal antibodies was performed by capture ELISA according to the instruction of SIGMA kit, which is as follows: diluting the monoclonal antibody subclass identification reagent 1:1000, adding into an enzyme-labeled hole, incubating at 100 mu L/hole for 1h at 37 ℃; PBST washing three times; diluting the antibody 1:1000 times, adding the sample, incubating at 37 ℃ for 1h at a concentration of 100 mu L/hole; PBST washing three times; diluting HRP enzyme-labeled goat anti-mouse IgG secondary antibody by 1:10000, adding the sample, incubating at 100 mu L/hole for 30min at room temperature; developing for 10-20 min. By OD₄₅₀The reading value is obviously higher than that of the subclass reagent added in other wells, namely the subclass type of the monoclonal antibody. The antibody subtype of the antibodies 1HD4 and 1JA9 is IgG 1.

2.2 measurement of antibody titer

The antibody titer after purification is measured by adopting an indirect ELISA method, and the method comprises the following steps: diluting MCR-1 protein to 0.2ug/mL and 100 ul/well, setting up a non-coating control, coating overnight at 4 ℃, spin-drying, and washing with PBST for 3 times; 5% of skim milk powder, 200 ul/hole, and sealing at 37 ℃ for 2 h; spin-dry, wash 3 times with PBST, add antibody (1 mg/ml concentration) diluted from 1:1000 times, add 12 gradients in total, set up uncoated control 100 ul/well at 37 deg.C for 1 h. Spin-dry, wash 3 times with PBST, add PBS 1:10000 times diluted goat anti-mouse second antibody, 100 ul/hole, 37 ℃, 45 min. Spin-drying, washing with PBST for 5 times, adding 100ul TMB/well, developing at 37 deg.C for 10min, terminating, and reading. The antibody is diluted to 1mg/ml after purification, and the titer reaches more than 1: 1280000.

2.3 antibody purity and molecular weight characterization

Performing antibody molecular weight and purity identification by an SDS-PAGE method; preparing glue, wherein the separation glue accounts for 12 percent, and the concentration glue accounts for 5 percent; preparing a sample, mixing 10ul of sample and 10ul of buffer, uniformly mixing, and boiling for 5 min; loading 10ul of sample in each hole, and setting up a protein pre-staining Marker contrast; 80V for 30min and 120V for 1.5 h; after electrophoresis, placing into a Coomassie brilliant blue solution for dyeing; decolorizing for 3 times (10 min each time); the molecular weight of the lgG antibody heavy chain is generally 50-75KDa, the molecular weight of the lgG antibody light chain is about 25KDa, and the purified monoclonal antibody is identified by SDS-PAGE; as shown in FIG. 2, antibodies 1HD4, 1JA9 each had clear bands at 50-75kDa and at about 25 kDa.

Example 3: gene verification of mouse anti-MCR-1 protein monoclonal antibody

Cloning Ig variable region gene by RT-PCR method, extracting total RNA, synthesizing single-chain cDNA. Total RNA from 1HD4 and 1JA9 hybridoma cell lines was extracted by Trizol (kit from Invitrogen) and reversed into a cDNA library by M-MLV reverse transcriptase (from Invitrogen).

Heavy chain framework region upstream primer

P1：5’SAGGTGMAGCTKCASSARTCWGG3’

Heavy chain variable region downstream primer

P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’

Light chain leader peptide upstream primer

P3：5’ATGGATTTTCAAGTGCAGATTTTCAG3’

Light chain variable region downstream primer

P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’

A PCR reaction system (50. mu.l) was prepared as follows:

2 μ l of cDNA; 2. mu.l of upstream primer (10. mu.M); 2. mu.l of downstream primer (10. mu.M); dNTP mix 2. mu.l; pfu DNA polymerase (5U/. mu.l) 1. mu.l; 10 Xpfu Buffer II: 5. mu.l; ddH₂Make up to 50. mu.l.

The reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; the following cycle was repeated 35 times: 30s at 95 ℃, 30s at 58 ℃ and 1min at 72 ℃; finally, extension was carried out at 72 ℃ for 10 min.

The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated to pMD19-T (sKPCle) vector (Takara) in the following manner:

VL PCR product/VH PCR product each 70ng, pMD19-T (sKPCle) vector 1. mu.l, Solution I ligation reaction Solution 5. mu.l; ddH₂O to 10. mu.l, ligation at 4 ℃Overnight.

The ligation products were transformed into E.coli DH5 alpha competent bacteria, cultured overnight at 37 ℃, single colonies were picked up, shaken at 37 ℃ for 2 hours, and then subjected to PCR identification of bacterial liquid, using cDNA of the corresponding antibody as a positive control. The reaction system (25. mu.l) was prepared as follows:

bacterial liquid: 1 μ l, forward primer (10 μ M): 1 mul; downstream primer (10 μ M): 1 mul; dNTP mix (2.5 Mm each) 2. mu.l; taq DNA polymerase (5U/. mu.l): 0.5 mul; 10 × Taq Buffer (Mg)²⁺plus): 2.5 mul; water was added to 25. mu.l. The reaction conditions were as before.

The PCR positive clone of the selected strain is expanded and cultured, and the positive clone plasmid is extracted by a plasmid extraction kit (Takara company) and is checked and sequenced. At least 5 clone samples were tested per chain of each antibody until the sequencing results were identical for at least three samples. The heavy chain and light chain variable region sequences of the antibodies 1HD4 and 1JA9 are obtained by successful cloning, and are matched with the variable region sequence characteristics of a typical antibody by alignment.

Example 4: enzyme linked immunosorbent assay (ELISA) reagent for detecting MCR-1 protein

Step one, preparing 1ml of HRP solution of 10mg/ml, and adding equal volume of 0.06M NaIO₄The reaction was carried out at 4 ℃ for 30 min. Adding 1ml of 0.16M ethylene glycol solution, standing in the shade for 30min, adding 10mg of 1JA9 antibody, adding into a 50KD dialysis bag, dialyzing in a carbonate buffer solution at 4 ℃ for two hours, taking out, adding 0.4ml of 5mg/ml sodium borohydride, reacting at 4 ℃ for 2 hours, adding equal volume of saturated ammonium sulfate, centrifuging, taking the precipitate PBS to redissolve to 2mg, dialyzing in a 200KD dialysis bag at 4 ℃ overnight, taking out, centrifuging, taking the supernatant, and fixing the volume to 5 ml;

step 1, diluting the antibody 1HD4 to 0.2mg/ml with PBS solution, coating each hole of an enzyme label plate with 100ul overnight at 4 ℃, adding 300ul of washing liquid into each hole, and washing for three times;

adding 5% of skim milk powder into PBST, placing an enzyme label plate at the temperature of 37 ℃ for sealing for 2h with each hole being 300ul, adding 300ul of washing liquid into each hole, and washing for three times;

step four, adding the HRP labeled antibody into a 96-well enzyme label plate fixed with the monoclonal antibody 1HD4, wherein each well has 50 ul. Diluting the antibody sample with antigen diluent, 50ul per well, acting at 37 ℃ for 1.5h, adding washing solution 300ul per well, and washing for four times;

step one, adding color development liquid into acanthopanax, incubating for 20min at 37 ℃ at 100 ul/hole;

step six stop solution was added at 50 ul/well, reading at 450nm wavelength and recording the results as in table 1.

TABLE 1

The antibody 1JA9 and the antibody 1HD4 can form a double-antibody sandwich, and the antigen detection sensitivity can reach 1:5120000 antibody dilution times.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.

Sequence listing

<110> Tianjin-Rui Biotechnology Ltd

Beijing Jinshan Chuan science and technology Co Ltd

TIANJIN XINUO BIOLOGICAL PHARMACEUTICAL Co.,Ltd.

<120> mouse anti-MCR-1 protein hybridoma cell strain, monoclonal antibody and application

<130> 2021

<160> 20

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Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

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Tyr Thr Ser Arg Leu His Ser

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Gln Gln Gly Asn Thr Phe Pro Tyr Thr

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Gly Tyr Ile Phe Thr Cys Cys Lys Met Tyr

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<213> Artificial Sequence (Artificial Sequence)

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Tyr Phe Asp Pro Tyr Asn Gly Asp Thr Ser Ser Asn Gln Lys Phe Lys

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Gly

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<213> Artificial Sequence (Artificial Sequence)

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Trp Leu Gln Asn Tyr Tyr Ala Met Asp Tyr

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Asp Val Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

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Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

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Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

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Arg Ser Ser Gln Asn Ile Val His Ser Tyr Gly Asn Pro Ser Leu Glu

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Gly

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ggacagggcc ttgagtggat tggatatttt aatccttaca ctgatggttc taagtacaat 180

gagatgttca acggcaaggc cacactgtcc tcagacaaat cctccggcac agcctatatg 240

gagttcagca gcctgacctc tgaggactct gcggtctatt actgtgcgag aggcccctat 300

ggtaactatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc cgcagccaaa 360

acg 363

Claims

1. The mouse MCR-1 protein hybridoma cell strain is characterized in that: is named as 1HD4 with the preservation number of CGMCC No. 23031; or 1JA9 with the preservation number of CGMCC No. 23033.

2. A murine anti-MCR-1 protein antibody characterized by: antibody 1HD4 comprising a light chain variable region comprising CDRL1 as shown in SEQ ID NO:1, CDRL2 as shown in SEQ ID NO:2 and CDRL3 as shown in SEQ ID NO:3 and a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO:4, CDRH2 as shown in SEQ ID NO:5 and CDRH3 as shown in SEQ ID NO: 6;

or,

antibody 1JA9, the light chain variable region comprising CDRL1 as shown in SEQ ID NO:11, CDRL2 as shown in SEQ ID NO:12 and CDRL3 as shown in SEQ ID NO:13, the heavy chain variable region comprising CDRH1 as shown in SEQ ID NO:14, CDRH2 as shown in SEQ ID NO:15 and CDRH3 as shown in SEQ ID NO: 16.

3. A murine anti-MCR-1 protein antibody according to claim 2 characterized by: the amino acid sequence of the light chain variable region of the antibody 1HD4 is shown as SEQ ID NO. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9;

or,

the amino acid sequence of the light chain variable region of the antibody 1JA9 is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 19.

4. A murine anti-MCR-1 protein antibody according to claim 2 or 3 characterized by: the antibody 1HD4 is produced by a mouse anti-MCR-1 protein hybridoma cell strain with the preservation number of CGMCC No. 23031;

5. A nucleic acid molecule, wherein: comprising nucleotides encoding a murine anti-MCR-1 protein antibody according to claim 2 or 3.

6. The nucleic acid molecule of claim 5, wherein: the nucleotide sequence of the variable region of the light chain of the nucleic acid molecule coding antibody 1HD4 is shown as SEQ ID NO. 8, and the nucleotide sequence of the variable region of the heavy chain of the nucleic acid molecule coding antibody 1HD4 is shown as SEQ ID NO. 10;

or,

the nucleotide sequence of the light chain variable region of the nucleic acid molecule coding antibody 1JA9 is shown as SEQ ID NO. 18, and the nucleotide sequence of the heavy chain variable region of the nucleic acid molecule coding antibody 1JA9 is shown as SEQ ID NO. 20.

7. Use of a murine anti-MCR-1 protein antibody according to any one of claims 2-4 for the preparation of a reagent for detecting MCR-1 protein.

8. The use of a murine anti-MCR-1 protein antibody according to claim 7 in the preparation of a reagent for detecting MCR-1 protein, characterized in that: the mouse anti-MCR-1 protein antibody is used for preparing an in vitro diagnosis kit or a microfluid chip, and the in vitro diagnosis kit is a colloidal gold immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme linked immunosorbent assay kit or a fluorescence immunoassay kit.

9. The use of a murine anti-MCR-1 protein antibody according to claim 7 in the preparation of a reagent for detecting MCR-1 protein, characterized in that: preparing an enzyme-linked immunoassay kit, wherein the antibody 1HD4 is a coating antibody, and the antibody 1JA9 is an HRP-labeled antibody;