CN114317455A - Mouse anti-MCR-1 protein hybridoma cell strain, monoclonal antibody and application - Google Patents

Abstract

The present invention provides mouse anti-MCR-1 protein hybridoma cell lines, monoclonal antibodies and applications thereof. The mouse hybridoma monoclonal antibody screening and RT-PCR method are used to clone Ig variable region genes to obtain stable secretion of mouse anti-MCR-1 protein. The hybridoma cell line of the antibody and its variable region sequence; through systematic evaluation, the mouse anti-MCR-1 protein antibody has good performance in all aspects, with a titer of more than 1:1280000, suitable for use as an immunodiagnostic reagent for Preparation of an in vitro diagnostic kit for polymyxin-resistant strains.

Description

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

technical field

The invention belongs to the technical field of antibody preparation, in particular to a mouse anti-MCR-1 protein hybridoma cell line, a monoclonal antibody and applications thereof.

Background technique

Polymyxin is a cationic polypeptide antibiotic that acts on the cell wall lipopolysaccharide of Gram-negative bacteria and destroys the bacterial cell structure. It is the most effective antibiotic for the treatment of carbapenem-resistant Gram-negative bacteria. In recent years, due to the increased clinical use of polymyxins, the number of drug-resistant strains has also increased year by year. Among the various drug mechanisms, mcr-1 belongs to the phosphoethanolamine transferase family. MCR-1 protein can modify lipid A, resulting in a decrease in the affinity of polymyxin for bacterial lipopolysaccharide, thereby mediating bacterial resistance to polymyxin. . At the same time, the mcr-1 gene spreads horizontally in different bacteria through plasmids, and can even co-exist with other drug resistance genes in the same plasmid, resulting in multiple drug resistance mechanisms after expression. Therefore, it is particularly important to establish a rapid and accurate method for the detection of mcr-1-resistant strains.

At present, the detection of mcr-1-resistant strains is mainly through molecular biology methods, but it is not easy to popularize due to its high requirements on detection equipment and environment. Immunological analysis methods have the advantages of high sensitivity, strong specificity, short cycle, simple operation and low cost, among which enzyme-linked immunosorbent assay is the most commonly used method. In view of the above situation, the present invention extracts MCR-1 protein as an antigen, and after obtaining a high-purity antigen, a good immune response is aroused in mice, and then hybridoma technology is used to screen high-affinity and specific monoclonal antibodies. On this basis, a double-antibody sandwich ELISA method was established to achieve rapid detection of mcr-1-resistant strains.

SUMMARY OF THE INVENTION

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

The technical scheme adopted in the present invention is: the mouse anti-MCR-1 protein hybridoma cell line is named 1HD4, and the deposit number is CGMCC No. 23031; or it is named 1JA9, and the deposit number is CGMCC No. 23033.

Mouse anti-MCR-1 protein antibody, antibody 1HD4, 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 and CDRL2 as 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 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)

or,

Antibody 1JA9, the light chain variable region includes 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, and the heavy chain variable region includes as SEQ ID NO : CDRH1 shown in 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)

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

SEQ ID NO: 7

DVQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGADYSLTISNLEEEDIATYFCQQGNTFPYTFGGGTKLEIK

SEQ ID NO: 9

VKLQESGPELVKPGASVKVSCKASGYIFTCCKMYWVKQSHGKSLEWIGYFDPYNGDTSSNQKFKGKVTLTVDKSSSTAYMFLNSLTSEDSAVYYCASWLQNYYAMDYWGQGTSVTVSS

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

SEQ ID NO: 17

EILLTQTPLSLPVSLGDQASISCRSSQNIVHSYGNPSLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHVPWTFGGGTKLEIKRAD

SEQ ID NO: 19

VKLQESGPELVKPGASVKMSCRTSGYTFTSYIMHWVKQKPGQGLEWIGYFNPYTDGSKYNEMFNGKATLSSDKSSGTAYMEFSSLTSEDSAVYYCARGPYGNYAMDYWGQGTSVTVSAAKT

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

Antibody 1JA9 was produced by a mouse anti-MCR-1 protein hybridoma cell line with deposit number 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 the 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 the antibody 1HD4 is shown in SEQ ID NO: 8 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 encoding antibody 1JA9 is shown in SEQ ID NO: 18, and the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody 1JA9 is shown in SEQ ID NO: 20 shown;

SEQ ID NO: 18

GAGATCTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTTATGGAAACCCCTCTTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGCTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT

SEQ ID NO: 20

GTGAAGCTTCAGGAATCTGGACCTGAGCTGGTTAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAGGACTTCTGGCTACACATTCACTAGCTATATTATGCACTGGGTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACACTGATGGTTCTAAGTACAATGAGATGTTCAACGGCAAGGCCACACTGTCCTCAGACAAATCCTCCGGCACAGCCTATATGGAGTTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCGAGAGGCCCCTATGGTAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGCAGCCAAAACG

The application of mouse anti-MCR-1 protein antibody in the preparation of MCR-1 protein detection reagent.

Preferably, the mouse anti-MCR-1 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 immunoassay kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immunoassay reagent kit or fluorescent immunoassay kit.

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

Alternatively, antibody 1JA9 is a coating antibody and antibody 1HD4 is an HRP-labeled antibody.

The advantages and positive effects of the present invention are as follows: the present invention provides two mouse anti-MCR-1 protein hybridoma cell lines, which can respectively produce two mouse anti-MCR-1 protein antibodies; In the evaluation of titer, kit sensitivity, specificity and stability, mouse anti-MCR-1 protein monoclonal antibody has good performance in all aspects, and the titer reaches more than 1:1280000, so it is suitable for use as an immunodiagnostic reagent for Preparation of an in vitro diagnostic kit for polymyxin-resistant strains.

Description of drawings

Fig. 1 is the electrophoresis image of mouse anti-MCR-1 protein;

Figure 2 is a protein electrophoresis image of mouse anti-MCR-1 protein antibody;

Biological material: 1HD4, the preservation date is September 24, 2021, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Management Committee (CGMCC), and the preservation number is CGMCC No.23031;

Biological material: 1JA9, the preservation date is September 24, 2021, the preservation unit is the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC), and the preservation number is CGMCC No.23033.

Detailed ways

Embodiments of the present invention will be described below.

The invention relates to a mouse anti-MCR-1 protein hybridoma cell line, the biological material is named 1HD4, belongs to the hybridoma cell line, and its preservation number is CGMCC No. 23031; Date is September 24, 2021, tested alive. Another mouse anti-MCR-1 protein hybridoma cell line, the biological material is named 1JA9, belongs to the hybridoma cell line, and its preservation number is CGMCC No. 23033; the preservation place is the General Microbiology Center of the China Microorganism Culture Collection Management Committee, whose preservation is Date is September 24, 2021, tested alive.

The antibody 1HD4 produced by the mouse anti-MCR-1 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, SEQ ID NO: CDRL2 shown in 2 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 shown in SEQ ID NO:6 CDRH3;

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

SEQ ID NO: 7

DVQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGADYSLTISNLEEEDIATYFCQQGNTFPYTFGGGTKLEIK

SEQ ID NO: 9

VKLQESGPELVKPGASVKVSCKASGYIFTCCKMYWVKQSHGKSLEWIGYFDPYNGDTSSNQKFKGKVTLTVDKSSSTAYMFLNSLTSEDSAVYYCASWLQNYYAMDYWGQGTSVTVSS

The nucleotide sequence encoding the light chain variable region of antibody 1HD4 is shown in SEQ ID NO: 8, and the nucleotide sequence 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

Antibody 1JA9 produced by mouse anti-MCR-1 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 shown in SEQ ID NO:13 CDRL3, the heavy chain variable region includes 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)

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

SEQ ID NO: 17

EILLTQTPLSLPVSLGDQASISCRSSQNIVHSYGNPSLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQASHVPWTFGGGTKLEIKRAD

SEQ ID NO: 19

VKLQESGPELVKPGASVKMSCRTSGYTFTSYIMHWVKQKPGQGLEWIGYFNPYTDGSKYNEMFNGKATLSSDKSSGTAYMEFSSLTSEDSAVYYCARGPYGNYAMDYWGQGTSVTVSAAKT

The nucleotide sequence encoding the light chain variable region of antibody 1JA9 is shown in SEQ ID NO: 18, and the nucleotide sequence encoding the heavy chain variable region of antibody 1JA9 is shown in SEQ ID NO: 20;

SEQ ID NO: 18

GAGATCTTGCTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTACATAGTTATGGAAACCCCTCTTTAGAGTGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGCTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGAT

SEQ ID NO: 20

GTGAAGCTTCAGGAATCTGGACCTGAGCTGGTTAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAGGACTTCTGGCTACACATTCACTAGCTATATTATGCACTGGGTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATTTTAATCCTTACACTGATGGTTCTAAGTACAATGAGATGTTCAACGGCAAGGCCACACTGTCCTCAGACAAATCCTCCGGCACAGCCTATATGGAGTTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCGAGAGGCCCCTATGGTAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCGCAGCCAAAACG

The mouse anti-MCR-1 protein monoclonal antibody has been systematically evaluated, including the evaluation of antibody subtype and titer, the sensitivity, specificity and stability of the kit. Therefore, it is suitable for the preparation of in vitro diagnostic kits as an immunodiagnostic reagent. It can be made into a colloidal gold immune kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immune kit or a fluorescent immune kit, or a microfluidic chip; the fabricated kit can detect MCR-1 protein. The two antibodies involved in this scheme are especially suitable for the formation of ELISA reagents. Antibody 1HD4 is a coating antibody, and antibody 1JA9 is an HRP-labeled antibody. The obtained ELISA reagent has higher sensitivity. 1HD4 was used as HRP-labeled antibody, and antibody 1JA9 was used as coating antibody.

The present invention will be further described below through specific embodiments. Among them, the experimental methods that do not specify the operation steps are all carried out according to the corresponding product instructions. The instruments, reagents, and consumables used in the examples can be purchased from commercial companies unless otherwise specified.

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

1.1 Antigen Preparation

Find and download the mcr-1 gene sequence from NCBI, and transform the recombinant plasmid into E. coli for inducible expression; the purification method is nickel column affinity chromatography, the bacteria are sonicated until the bacteria liquid is clarified, high-speed centrifugation, and the supernatant is filtered through a membrane After passing through the nickel column, the target protein was eluted, and the molecular weight was consistent with the expected 40.6KDa. See Figure 1 for SDS-PAGE and quantify by BCA method.

1.2 Mice immunization

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, the mice were divided into 2 groups with 3 mice in each group; according to the antigen content, the immunization dose of the first group was 25ug/mouse, the second group of immunization dose is 50ug/mouse, first immunization, take an appropriate amount of MCR-1 protein and dilute it with normal saline to 500ul, add an equal amount of Freund's complete adjuvant 500ul to emulsify evenly, and subcutaneously inject immunized mice at multiple points; Two weeks later, the same dose was taken for the second immunization, the adjuvant was changed to Freund's incomplete adjuvant, and the mice were immunized by intraperitoneal injection. After two weeks, the mice were immunized again by intraperitoneal injection. Blood was collected from the tail of the mice, and the serum titers of the mice were determined by ELISA.

The specific steps are as follows: MCR-1 protein 0.2ug/ml, 100ul/well, coat ELISA plate overnight at 4°C, spin dry, and wash 3 times with PBST. 5% skim milk powder, 250ul/well, closed for 2h. Blood was collected from the mouse tail, 3000 RPM, and the serum was collected after centrifugation. From 1:1000, it was diluted to 1:5120000 with PBS, and it was used for later use. Spin dry, wash 3 times with PBST, add primary antibody diluted in PBS, 100ul/well, 37°C, 1h. Wash 3 times with PBST, add goat anti-mouse secondary antibody, 100ul/well, 37 degrees, 40min. Wash 4 times with PBST, add 100ul TMB/well, 37°C, develop color for 20min, stop, and read the value.

1.3 Cell fusion

Three days before fusion, mice were boosted with the same amount of inoculation as the previous immunization, without adjuvant, intraperitoneal injection. The feeder cells were prepared one day before fusion, and one Balb/c mouse, 6-8 weeks old, was taken and killed by cervical dislocation after the eyeball was exsanguinated. It was sterilized in 75% alcohol, and the abdominal skin was aseptically cut in an ultra-clean bench. Inject 10 ml of the HAT selection medium into the abdominal cavity of the mouse, rub the abdomen lightly with an alcohol cotton ball, and withdraw the medium. Add 40ml of HAT medium, spread into 4 96-well cell culture plates, 100μL/well, 37°C, 5% CO ₂ cell culture incubator. Myeloma cells (Sp2/0 cells) were recovered one week before fusion, cultured in PRMI-1640 medium containing 10% fetal bovine serum, and subcultured in a 37°C, 5% CO ₂ incubator. The cells in the logarithmic growth phase were collected into a centrifuge tube, counted, and the cells were diluted to 10 ⁷ cells/ml for use. The Balb/c mice that had been boosted for 3 days were taken, the eyeballs were removed for bloodletting to prepare positive serum, the cervical vertebrae were sacrificed, and the 75% alcohol was disinfected for 5 min. connective tissue. Put the spleen on the microporous copper mesh, add fresh RPMI-1640 culture medium, first use a syringe to suck the culture medium and inject it from the spleen section, blow off the spleen cells, after repeated several times, use the inner plug of the syringe to gently grind the remaining spleen. , until there is no obvious red tissue mass. The spleen cell suspension in the plate was gently pipetted and then transferred to a 50 ml centrifuge tube, centrifuged at 1000 r/min for 5 min, and the spleen cells were collected and counted for later use. The immunized mouse splenocytes and Sp2/0 cells were mixed at a ratio of 10:1 according to the number of cells, added to a 50 ml centrifuge tube, centrifuged at 1000 r/min for 5 min, the supernatant was discarded, and the two cells were thoroughly mixed by rubbing gently in the palm of the hand. As for the 100mL blue-capped bottle, the blue-capped bottle is filled with 37°C hot water, and 1ml of preheated DMSO/PEG is added dropwise to the fusion tube within 1min. Slowly then fast, while adding, gently rotate the centrifuge tube. Then immediately add anti-blood and blood-free RPMI-1640 culture medium to stop the reaction, add 1 ml in the first minute, add 2 ml in the second minute, add 3 ml in the third minute, and add 4 ml in the fourth minute. 37 °C water bath for 5 min, then centrifuged at 800 r/min for 5 min, discard the supernatant, suspend the pellet with HAT, mix well into 40 ml of HAT selection medium containing 20% calf serum preheated at 37 °C, and add Feed cells into a 96-well cell plate, 100 μL/well, and place the culture plate into a 37°C, 5% CO ₂ incubator. After 7 days, the medium of the cell plate was half-changed with fresh HAT medium, and the medium was completely changed with HT medium after 10 days. The positive cells in the 96-well plate were subcloned by the limiting dilution method: first, prepare feeder cells according to the above method, take the hybridoma cells to be cloned for cell counting, and dilute the cells with HT medium to 5-8 cells/ ml, add 100 μL/well to a 96-well cell plate that has been plated with feeder cells, clone a 96-well cell plate for each hybridoma cell, and culture in a 37°C, 5% CO ₂ cell incubator. After about 5 days, the number of clones in the cell wells was counted, marked, and replaced with a new medium after 7 days, and the cells were detected when the cells covered 1/3 to 1/2 of the entire bottom of the well. After 2-3 times of cloning, when all the cell wells of the 96-well plate are positive, it can be expanded, cultured, established, and frozen. Hybridoma cells confirmed by positive test were expanded for culture and cryopreserved. The specific process is as follows: the vigorously growing hybridoma cells in good condition are gently blown off the cell flask with anti-blood-free DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant is discarded. Add freezing solution (containing 40% RPMI-1640 culture medium, 50% fetal bovine serum, 10% DMSO), blow off the cells and distribute them into cell cryopreservation tubes. Put the cryovials in a freezing box and place them in a -70°C freezer. One day later, transfer the cryovials to liquid nitrogen and make a record.

1.4 Preparation of ascites

10-12-week-old female Balb/c mice were injected intraperitoneally with sterile liquid paraffin, 0.5 mL/mice, and 7 days later, hybridoma cells cultured to log phase were intraperitoneally injected, 5×10 ⁶ cells/mice. Pay attention to observation every day, about 7-10 days, after the abdomen of the mouse has obvious bulge, sterilize the lower abdominal skin with a 75% alcohol cotton ball, pierce the abdominal cavity with a needle, and collect ascites. After the ascites regenerated and accumulated, it was collected again. The collected ascites was centrifuged at 3000 r/min for 10 min, and the middle clarified part was taken, filtered with filter paper, packaged and stored at -70°C.

1.5 Antibody purification

Purify ascites with Protein-G column. The steps are as follows: take 2ml (n) of ascites, centrifuge at 10,000g, take the clear part, add 2ml (1:1) washing buffer, mix well, drain the column with 20% ethanol, and then use 8mL The washing solution is balanced, the sample is passed through the column, the flow rate is 8S/drop, and the sample is loaded repeatedly 3 times, and then the precipitate is washed with 15mL washing buffer, and the flow rate is 8S/drop. After washing, wash with 10mL elution buffer. After elution, the pH will be adjusted to 7.4 with 1M Tris PH=9, then concentrated with a concentration column, and dialyzed overnight at 50kd dialysis bag, PBS, and 4°C.

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

2.1 Identification of antibody subclasses

According to the instructions of the SIGMA kit, the subclass identification of monoclonal antibodies was carried out by the capture ELISA method. The details are as follows: after diluting the subclass identification reagent of monoclonal antibody at 1:1000, add it to the enzyme label well, 100μL/well, and incubate at 37°C for 1h; Wash three times with PBST; add the antibody after 1:1000 dilution, 100 μL/well, incubate at 37°C for 1 h; wash three times with PBST; HRP enzyme-labeled goat anti-mouse IgG secondary antibody is diluted 1:10000 and add the sample, 100 μL/well , incubate for 30min at room temperature; develop color for 10-20min. The OD ₄₅₀ reading value was significantly higher than that of the subtype reagents added in other wells as the subtype of the monoclonal antibody. The antibody subtype of antibodies 1HD4 and 1JA9 is IgG1.

2.2 Antibody titer determination

The indirect ELISA method was used to determine the antibody titer after purification. The steps were as follows: Dilute MCR-1 protein to 0.2ug/mL, 100ul/well, set up an uncoated control, coat overnight at 4°C, spin dry, and wash 3 times with PBST ; 5% nonfat milk powder, 200ul/well, closed at 37 degrees for 2h; dried, washed 3 times with PBST, and added the antibody (concentration is 1mg/ml) from 1:1000 times for doubling dilution, a total of 12 gradients, At the same time, set up uncoated control 100ul/well, 37℃, 1h. Spin dry, wash 3 times with PBST, add goat anti-mouse secondary antibody diluted 1:10000 times in PBS, 100ul/well, 37°C, 45min. Spin dry, wash 5 times with PBST, add 100ul TMB/well, 37°C, develop color for 10min, stop, and read the value. After purification, the antibody was diluted to 1mg/ml, and the titer reached more than 1:1,280,000.

2.3 Antibody purity and molecular weight identification

SDS-PAGE method was used to identify the molecular weight and purity of the antibody; for gel preparation, the separation gel was 12%, and the stacking gel was 5%; sample preparation, 10ul sample + 10ul buffer, mixed well, boiled for 5min; 10ul sample was loaded in each well, and set up at the same time Protein pre-stained Marker control; 80 volts for 30 minutes, 120 volts for 1.5 hours; after electrophoresis, put it into Coomassie brilliant blue solution for staining; de-stain, boil and de-ionized water for 10 minutes, a total of 3 times; IgG antibody heavy chain molecules The mass is generally 50-75KDa, and the molecular weight of the IgG antibody light chain is about 25KDa. The purified monoclonal antibody was identified by SDS-PAGE; as shown in Figure 2, antibodies 1HD4 and 1JA9 have 50-75KDa and about 25KDa respectively. Clear bands.

Example 3: Gene validation of mouse anti-MCR-1 protein monoclonal antibody

Ig variable region gene was cloned by RT-PCR, total RNA was extracted, and single-stranded cDNA was synthesized. The total RNA of 1HD4 and 1JA9 hybridoma cell lines was extracted by Trizol method (purchased from Invitrogen), and the total RNA was reversed into cDNA library with M-MLV reverse transcriptase (purchased from Invitrogen).

Primer upstream of heavy chain backbone region

P1: 5'SAGGTGMAGCTKCASSARTCWGG3'

Heavy chain variable region downstream primers

P2: 5'TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3'

Light chain leader peptide upstream primer

P3: 5'ATGGATTTTCAAGTGCAGATTTTCAG3'

Light chain variable region downstream primer

P4: 5'GGATACAGTTGGTGCAGCATCAGCCCGTTT3'

The PCR reaction system (50 μl) was prepared as follows:

cDNA: 2 μl; upstream primer (10 μM): 2 μl; downstream primer (10 μM): 2 μl; dNTP mixture: 2 μl; pfuDNA polymerase (5U/μl): 1 μl; 10×pfu Buffer II: 5 μl; ddH ₂ O: make up to 50μl.

Reaction conditions: pre-denaturation at 95 °C for 5 min; repeat the following cycle 35 times: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min; finally, extension at 72 °C for 10 min.

The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments are respectively connected with pMD19-T (sKPCle) vector (Takara company), and the connection system is as follows:

VL PCR product/VH PCR product each 70ng, pMD19-T(sKPCle) vector 1μl, Solution I ligation reaction solution 5μl; ddH ₂ O supplemented to 10μl, ligation overnight at 4°C.

The ligation product was transformed into E.coli DH5α competent bacteria, and after overnight incubation at 37°C, a single colony was picked, shaken at 37°C for 2 hours, and then identified by bacterial liquid PCR. The cDNA of the corresponding antibody was used as a positive control. The reaction system (25 μl) was prepared as follows:

Bacterial solution: 1 μl, upstream primer (10 μM): 1 μl; downstream primer (10 μM): 1 μl; dNTP Mixture (2.5 Mm each) 2 μl; Taq DNA polymerase (5U/μl): 0.5 μl; 10×Taq Buffer ( Mg ²⁺ plus): 2.5 μl; make up to 25 μl with water. The reaction conditions were the same as before.

The PCR-positive clones of the bacteria were selected for expansion and culture, and the positive cloned plasmids were extracted with a plasmid extraction kit (Takara Company), and sent to the detection sequence. At least 5 clone samples were submitted for each chain of each antibody until at least three samples had the same sequencing result. The variable region sequences of the heavy chain and light chain of antibodies 1HD4 and 1JA9 were successfully cloned, which conformed to the characteristics of typical antibody variable region sequences after comparison.

Example 4: Detection of MCR-1 protein by enzyme-linked immunosorbent assay

Step 1: Prepare 1 ml of 10 mg/ml HRP solution, add an equal volume of 0.06 M NaIO ₄ , and react at 4° C. for 30 min. Then add 1ml of 0.16M ethylene glycol solution, place in the dark for 30min, add 10mg 1JA9 antibody, add it to a 50KD dialysis bag, dialyze it in carbonate buffer at 4°C for two hours, take out and add 0.4ml of 5mg/ml sodium borohydride, 4 React at ℃ for 2h, add equal volume of saturated ammonium sulfate, centrifuge and redissolve the precipitate in PBS to 2mg, dialyze overnight at 4℃ in a 200KD dialysis bag, take out the centrifuge to take the supernatant, and dilute to 5ml;

Step two: The antibody 1HD4 was diluted to 0.2mg/ml with PBS solution, 100ul per well of the ELISA plate was coated overnight at 4°C, 300ul/well of washing solution was added, and washed three times;

Step 3: Add 5% skim milk powder to PBST, 300ul per well of the ELISA plate, place at 37°C for blocking for 2h, add 300ul/well of washing solution, and wash three times;

Step 4 Add the HRP-labeled antibody to the 96-well ELISA plate immobilized with the monoclonal antibody 1HD4, 50ul per well. Dilute the antibody sample with antigen diluent, 50ul per well, place it at 37°C for 1.5h, add 300ul/well of washing solution, and wash four times;

Step 5: Add chromogenic solution, 100ul/well, incubate at 37°C for 20min;

Step 6: Add stop solution, 50ul/well, read at the wavelength of 450nm, and record the results as shown in Table 1.

Table 1

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

The embodiments of the present invention have been described in detail above, but the above contents are only preferred embodiments of the present invention, and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made according to the scope of the application of the present invention should still belong to the scope of the patent of the present invention.

sequence listing

<110> Tianjin Yirui Biotechnology Co., Ltd.

Beijing Jinshanchuan Technology Co., Ltd.

Tianjin Xinuo Biopharmaceutical Co., Ltd.

<120> Mouse anti-MCR-1 protein hybridoma cell line, monoclonal antibody and application

<130> 2021

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Tyr Thr Ser Arg Leu His Ser

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acg 363

Claims (9)

1. A mouse anti-MCR-1 protein hybridoma cell line, characterized in that: it is named 1HD4, and the deposit number is CGMCC No. 23031; or it is named 1JA9, and the deposit number is CGMCC No. 23033. 2. mouse anti-MCR-1 protein antibody, it is characterized in that: antibody 1HD4, comprises light chain variable region and heavy chain variable region, and light chain variable region comprises CDRL1 as shown in SEQ ID NO:1, SEQ ID NO CDRL2 shown in: 2 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 shown in SEQ ID NO: 6 CDRH3; or, Antibody 1JA9, the light chain variable region includes 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, and the heavy chain variable region includes as SEQ ID NO : CDRH1 shown in SEQ ID NO: 14, CDRH2 shown in SEQ ID NO: 15, and CDRH3 shown in SEQ ID NO: 16. 3. mouse anti-MCR-1 protein antibody according to claim 2 is characterized in that: antibody 1HD4 light chain variable region amino acid sequence is shown in SEQ ID NO: 7, heavy chain variable region amino acid sequence is SEQ ID NO :9 shown; or, The amino acid sequence of the variable region of the light chain of antibody 1JA9 is shown in SEQ ID NO: 17, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 19. 4. The mouse anti-MCR-1 protein antibody according to claim 2 or 3, wherein the antibody 1HD4 is produced by the mouse anti-MCR-1 protein hybridoma cell line with the deposit number of CGMCC No.23031; Antibody 1JA9 was produced by the mouse anti-MCR-1 protein hybridoma cell line with deposit number CGMCC No. 23033. 5. A nucleic acid molecule, characterized in that it comprises a nucleotide encoding the mouse anti-MCR-1 protein antibody of claim 2 or 3. 6. The nucleic acid molecule according to claim 5, wherein the nucleic acid molecule encodes the nucleotide sequence of the light chain variable region of the antibody 1HD4 as shown in SEQ ID NO: 8, and the nucleic acid molecule encodes the antibody 1HD4 The nucleotide sequence of the variable region of the heavy chain is shown in SEQ ID NO:10; or, The nucleotide sequence of the light chain variable region of the nucleic acid molecule encoding antibody 1JA9 is shown in SEQ ID NO: 18, and the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody 1JA9 is shown in SEQ ID NO: 20 shown. 7. The application of the mouse anti-MCR-1 protein antibody according to any one of claims 2-4 in the preparation of a reagent for detecting MCR-1 protein. 8. the application of the mouse anti-MCR-1 protein antibody according to claim 7 in the preparation and detection of the MCR-1 protein reagent, it is characterized in that: the mouse anti-MCR-1 protein antibody is used to prepare in vitro diagnostic kits or microfluidics A chip, wherein the in vitro diagnostic kit is a colloidal gold immune kit, a chemiluminescence kit, a radioimmunoassay kit, an enzyme-linked immune kit or a fluorescence immune kit. 9. the application of the mouse anti-MCR-1 protein antibody according to claim 7 in the preparation and detection of MCR-1 protein reagent, it is characterized in that: preparing an enzyme-linked immunosorbent assay kit, antibody 1HD4 is a coating antibody, and antibody 1JA9 is HRP labeled antibody; Alternatively, antibody 1JA9 is a coating antibody and antibody 1HD4 is an HRP-labeled antibody.

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