CN114773456B - anti-PK 34 monoclonal antibody and hybridoma cell line as well as preparation and purification methods thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-PK 34 monoclonal antibody and a hybridoma cell line as well as preparation and sequencing methods thereof. The preservation number of the hybridoma cell line is CCTCC NO: C202226. The preparation method comprises the following steps: PK34 as an immunogen to immunize mice, the potency of antisera to PK34 was determined by ELISA; preparing trophoblasts, myeloma cells and splenocytes, electrically fusing the cells, and screening to obtain a fused cell strain specific to PK 34; and (3) performing cell subcloning, and screening to obtain a monoclonal cell strain with higher specificity. The anti-PK 34 monoclonal antibody and the hybridoma cell line thereof are prepared, and full-length sequencing of the hybridoma antibody is completed. The recombinant antibody can be obtained in vitro through genetic engineering in the follow-up process, so that the risk of degradation positive loss of hybridoma cells is avoided greatly. If the PK34 antibacterial peptide can be applied to the clinical treatment of tuberculosis in the future, the antibody can be used for analyzing diseases and monitoring conditions.

Description

anti-PK 34 monoclonal antibody and hybridoma cell line as well as preparation and purification methods thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-PK 34 monoclonal antibody and a hybridoma cell line as well as preparation and purification methods thereof.

Background

Tuberculosis is an infectious disease that severely jeopardizes public health, and the pathogen is mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB). Although tuberculosis can be prevented and cured by chemical drugs, because the chemotherapeutic drugs have short plates with strong toxic and side effects, and the anti-tuberculosis treatment time is long and the combination of a plurality of chemotherapeutic drugs causes the problem that MTB is easy to have multi-drug resistance and wide drug resistance, the development of a novel MTB infection resistance and focus repair method has important value.

The antibacterial peptide as a biological antibacterial drug has a series of advantages: has wide application, broad antibacterial spectrum, rapid sterilization, no accumulation and poisoning effects, etc. The antibacterial peptide has different action mechanisms from the traditional antibacterial drugs, so that the antibacterial peptide becomes an important candidate object for resisting drug-resistant bacteria. Studies have shown that antibacterial peptides are effective in treating tuberculosis and have no adverse reactions such as liver function damage, gastrointestinal symptoms, anaphylaxis and the like of tuberculosis chemotherapeutic drugs. Researchers such as Wei select a small molecule polypeptide PK34 from MTB phage D29, and can specifically bind to the most abundant sugar ester on the surface of MTB, namely Trehalose-di-mycolate (TDM). TDM is a surface core molecule of MTB, an important target for developing anti-MTB drugs. PK34 has both the ability to kill MTB and anti-inflammatory responses. In vivo experiments showed that PK34 had an ability to clear MTB comparable to rifampicin. PK34 inhibits the massive secretion of inflammatory factors by inhibiting the activation of mitogen-activated protein kinases and protein kinase B, but at the same time maintains certain inflammatory cytokine levels to maintain normal immunocompetence. PK34 is expected to be an adjunct to the treatment of MTB infection or as a template for the development of antitubercular drugs.

No monoclonal antibodies against PK34 polypeptides have been published at present, mainly because polypeptides have a small molecular weight as immunogens and it is difficult to form mature B cells with secretion specific antibodies in secondary lymphoid organs in mice. Furthermore, the screening of cell clones after obtaining stable cell clones with antibody secretion function is a cumbersome and time-consuming process for the identification of secreted antibodies. In addition, due to the characteristic of polyploidy, the hybridoma cells can cause mutation or genetic drift after long-term storage, so that antibodies are not generated or nonspecific antibodies are generated, the obtained hybridoma cells have genetic instability, and the risk of degradation positive loss of the hybridoma cells is easy to occur. Secondly, if the PK34 antibacterial peptide can be applied to clinical treatment of tuberculosis in the future, the PK34 monoclonal antibody is used for constructing an in vitro immunological technical method to determine the protein level concentration, thereby being more beneficial to analysis and condition monitoring of diseases. In cell therapy, the corresponding gene of the PK34 polypeptide can be modified into cells, and the monoclonal antibody of the anti-PK 34 polypeptide can also be used for detecting the expression quantity of the PK34 in vivo.

Therefore, it is necessary to prepare a monoclonal antibody against the PK34 polypeptide and a hybridoma cell line against the PK34 monoclonal antibody, and sequence the hybridoma antibody gene of the hybridoma cell to obtain an antibody gene sequence. The recombinant antibody can be obtained easily in vitro in a genetic engineering mode, and antibody engineering projects such as antibody humanization and the like can be realized.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an anti-PK 34 monoclonal antibody and a hybridoma cell line as well as preparation and purification methods thereof, which are used for solving the problems related to the background art.

In order to achieve the above purpose, the present invention adopts the following technical scheme: a hybridoma cell line of anti-PK 34 monoclonal antibody, said hybridoma cell line having a accession number of cctccc No. c202226, class designation: a hybridoma cell line which is resistant to PK34 monoclonal antibodies. Preservation unit: china center for type culture Collection, address: the preservation date of the university of Wuhan in China is 2022, 1 month and 26 days.

According to a further aspect of the invention, the hybridoma cell line secretes a monoclonal antibody against PK 34.

The invention also comprises a preparation method of the hybridoma cell line of the PK34 monoclonal antibody, wherein the hybridoma cell line is the hybridoma cell line, and the preparation method comprises the following steps:

step 1: PK34 is used as an immunogen, mice are immunized, blood is taken and detected, and the titer of antisera against the PK34 is determined by an indirect ELISA method;

step 2: preparing trophoblasts, myeloma cells and splenocytes, performing cell electrofusion, and screening to obtain a fused cell strain with specificity for PK 34;

step 3: and (3) performing cell subcloning, and screening to obtain a monoclonal cell strain with higher specificity for PK 34.

According to a further technical scheme of the invention, the condition of cell electrofusion in the step 2 is that the alternating current calibration voltage is about 35V, the alternating current duration time is within 60s, the low voltage mode direct current fusion voltage is about 260V, and the direct current pulse width is about 40 mu s.

According to a further technical scheme, the specific steps of cell electrofusion in the step 2 are as follows:

(1) Mixing myeloma cells and spleen cells such that the myeloma cell to spleen cell number ratio is 1:2;

(2) Placing the cells into a centrifuge tube, diluting with DMEM basal medium, centrifuging at 1500rpm for 5min, and discarding the supernatant;

(3) Adding electrofusion solution into a centrifuge tube, shaking the centrifuge tube to make cells uniform, centrifuging at 1500rpm for 5min, discarding the supernatant, and washing the cells once;

(4) Then adding electrofusion solution to adjust the cell density to 2×10 ⁷ CFU/mL, then adding 2mL of cell suspension into the fusion pool for electrofusion on-machine experiment;

(5) Setting an alternating current calibration voltage of about 35V, an alternating current duration of less than 60s, a low voltage mode direct current fusion voltage of about 260V, and a direct current pulse width of about 40 mu s;

(6) Standing for 10min after electrofusion, centrifuging the fused cells at 1000rpm for 5min, collecting the supernatant, suspending the fused cells again with HAT DMEM medium, and adding the fused cells into a 96-well cell culture plate containing trophoblast prepared 1-2 days in advance, wherein the concentration of the fused cells is 100 mu L/well;

(7) The cell culture plates were then placed at 37℃in 5% CO ₂ Culturing in a cell culture box.

The invention also comprises a monoclonal antibody against PK34, which is secreted by the hybridoma cell line, or the hybridoma cell line is prepared by the method, wherein the heavy chain variable region gene sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region gene sequence of the monoclonal antibody is shown as SEQ ID NO. 2.

According to a further technical scheme, the polypeptide sequence encoded by the heavy chain variable region gene is shown as SEQ ID NO.3, and the polypeptide sequence encoded by the light chain variable region gene is shown as SEQ ID NO. 4.

According to a further technical scheme, the heavy chain signal peptide gene sequence of the monoclonal antibody is shown as SEQ ID NO.5, and the light chain signal peptide gene sequence is shown as SEQ ID NO. 6;

the polypeptide sequence coded by the heavy chain signal peptide gene is shown as SEQ ID NO.7, and the polypeptide sequence coded by the light chain signal peptide gene is shown as SEQ ID NO. 8.

According to a further technical scheme, the heavy chain constant region gene sequence of the monoclonal antibody is shown as SEQ ID NO.9, and the light chain constant region gene sequence is shown as SEQ ID NO. 10;

the polypeptide sequence of the heavy chain constant region gene is shown as SEQ ID NO.11, and the polypeptide sequence of the light chain constant region gene is shown as SEQ ID NO. 12.

The sequencing step of the monoclonal antibody comprises the following steps:

step 1: total RNA is extracted by hybridoma cell lysis:

step 2: reverse transcription of RNA into cDNA is carried out by using RACE technology, and then the full length of heavy chain and light chain is obtained by PCR amplification by taking cDNA as a template:

step 3: ligating the target fragment to the vector using a ligase and transforming the ligation product into E.coli competent cells, and then selecting a monoclonal for screening sequencing:

step 4: sequencing result analysis and annotation:

the invention also includes a method for purifying the anti-PK 34 monoclonal antibody,

step 1: antibody expression is carried out on the monoclonal cell strain;

step 2: slowly loading the cell supernatant by using an affinity purification chromatographic column, and eluting with glycine elution buffer after the antibody is combined to obtain the required purified antibody;

step 3: the antibodies were dialyzed overnight at 4℃in PBS, and purity and concentration were measured every other day.

The anti-PK 34 monoclonal antibody, the purification method, the hybridoma cell line and the preparation method have the advantages that: the invention prepares the anti-PK 34 monoclonal antibody and the hybridoma cell line thereof. If the PK34 antibacterial peptide can be applied to clinical treatment of tuberculosis in the future, the PK34 monoclonal antibody construction in vitro immunological technology method is utilized to determine the protein level concentration, which is more beneficial to analysis and condition monitoring of diseases. In cell therapy, the PK34 polypeptide corresponding gene can be modified into cells, and the anti-PK 34 polypeptide monoclonal antibody can also be used for detecting the expression quantity of PK34 in vivo, so that the research progress of tuberculosis is further promoted.

Drawings

FIG. 1 is an analysis chart of SDS-PAGE of an antibody according to an embodiment of the present invention;

FIG. 2 is a graph showing the WB assay results of PK34 with the antibodies of the examples of the present invention.

Detailed Description

The following description of the embodiments is provided in conjunction with the accompanying drawings.

The reagents and apparatus in the examples below were conventional experimental reagents and apparatus.

Example 1:

a hybridoma cell line of anti-PK 34 monoclonal antibody, wherein the preservation number of the hybridoma cell line is CCTCC NO: C202226.

According to a further aspect of the invention, the hybridoma cell line secretes a monoclonal antibody against PK 34.

The invention also comprises a preparation method of the hybridoma cell line of the PK34 monoclonal antibody, wherein the hybridoma cell line is the hybridoma cell line, and the preparation method comprises the following steps:

step 1: PK34 is used as an immunogen, mice are immunized, blood is taken and detected, and the titer of antisera against the PK34 is determined by an indirect ELISA method;

step 2: preparing trophoblasts, myeloma cells and splenocytes, performing cell electrofusion, and screening to obtain a fused cell strain with specificity for PK 34;

step 3: and (3) performing cell subcloning, and screening to obtain a monoclonal cell strain with higher specificity for PK 34.

According to a further technical scheme of the invention, the condition of cell electrofusion in the step 2 is that the alternating current calibration voltage is about 35V, the alternating current duration time is within 60s, the low voltage mode direct current fusion voltage is about 260V, and the direct current pulse width is about 40 mu s.

According to a further technical scheme, the specific steps of cell electrofusion in the step 2 are as follows:

(1) Mixing myeloma cells and spleen cells so that the number ratio of myeloma cells to spleen cells is 1;2;

(2) Placing the cells into a centrifuge tube, diluting with DMEM basal medium, centrifuging at 1500rpm for 5min, and discarding the supernatant;

(3) Adding electrofusion solution into a centrifuge tube, shaking the centrifuge tube to make cells uniform, centrifuging at 1500rpm for 5min, discarding the supernatant, and washing the cells once;

(4) Then adding electrofusion solution to adjust the cell density to 2×10 ⁷ CFU/mL, then adding 2mL of cell suspension into the fusion pool for electrofusion on-machine experiment;

(5) Setting an alternating current calibration voltage of about 35V, an alternating current duration of less than 60s, a low voltage mode direct current fusion voltage of about 260V, and a direct current pulse width of about 40 mu s;

(6) Standing for 10min after electrofusion, centrifuging the fused cells at 1000rpm for 5min, collecting the supernatant, suspending the fused cells again with HAT DMEM medium, and adding the fused cells into a 96-well cell culture plate containing trophoblast prepared 1-2 days in advance, wherein the concentration of the fused cells is 100 mu L/well;

(7) The cell culture plates were then placed at 37℃in 5% CO ₂ Culturing in a cell culture box.

The invention also comprises a monoclonal antibody against PK34, which is secreted by the hybridoma cell line, or the hybridoma cell line is prepared by the method, wherein the heavy chain variable region gene sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region gene sequence of the monoclonal antibody is shown as SEQ ID NO. 2.

The polypeptide sequence of the heavy chain variable region gene is shown as SEQ ID NO.3, and the polypeptide sequence of the light chain variable region gene is shown as SEQ ID NO. 4.

The heavy chain signal peptide gene sequence of the monoclonal antibody is shown as SEQ ID NO.5, and the light chain signal peptide gene sequence is shown as SEQ ID NO. 6;

the polypeptide sequence coded by the heavy chain signal peptide gene is shown as SEQ ID NO.7, and the polypeptide sequence coded by the light chain signal peptide gene is shown as SEQ ID NO. 8.

The heavy chain constant region gene sequence of the monoclonal antibody is shown as SEQ ID NO.9, and the light chain constant region gene sequence is shown as SEQ ID NO. 10;

the polypeptide sequence of the heavy chain constant region gene is shown as SEQ ID NO.11, and the polypeptide sequence of the light chain constant region gene is shown as SEQ ID NO. 12.

The sequencing step of the monoclonal antibody comprises the following steps:

step 1: total RNA is extracted by hybridoma cell lysis:

step 2: reverse transcription of RNA into cDNA is carried out by using RACE technology, and then the full length of heavy chain and light chain is obtained by PCR amplification by taking cDNA as a template:

step 3: ligating the target fragment to the vector using a ligase and transforming the ligation product into E.coli competent cells, and then selecting a monoclonal for screening sequencing:

step 4: sequencing result analysis and annotation:

the invention also includes a method for purifying the anti-PK 34 monoclonal antibody,

step 1: antibody expression is carried out on the monoclonal cell strain;

step 2: slowly loading the cell supernatant by using an affinity purification chromatographic column, and eluting with glycine elution buffer after the antibody is combined to obtain the required purified antibody;

step 3: the antibodies were dialyzed overnight at 4℃in PBS, and purity and concentration were measured every other day.

Example 2:

a preparation method of a hybridoma cell line of an anti-PK 34 monoclonal antibody,

step 1: animal immunization:

5 BALB/c mice were selected, PK34 and Freund's adjuvant were mixed and emulsified in a volume ratio of 1:1, and were immunized by subcutaneous injection at 50. Mu.g/dose, and boosted every 2-3 weeks. Wherein, the primary immunization is that PK34 is mixed with Freund's complete adjuvant, and the booster immunization is that PK34 is mixed with Freund's incomplete adjuvant. Blood collection assay, the titers of antisera against PK34 were determined by indirect ELISA.

Antiserum potency detection and screening:

(1) The coated plate was designed according to the experimental requirements and the strip was marked.

(2) PK34 was diluted to the required concentration of 1. Mu.g/mL with phosphate buffer (PBS, pH 7.4) as coating buffer, mixed well and added to the strip at 100. Mu.L per well overnight in a refrigerator at 4 ℃.

(3) After the coating, the coating solution was discarded, the plate was washed 3 times, 200. Mu.L of a blocking solution was added to each well, and the incubator at 37℃was operated for 1 hour. And taking out the ELISA plate, discarding the internal liquid, and washing the plate for 1 time.

(4) Antisera were diluted 1:500, 3-fold, 100. Mu.L per well, incubator at 37℃for 1h.

(5) Taking out the ELISA plate, discarding the inner liquid, washing the plate for 3 times, and adding 100 mu L of diluted ELISA secondary antibody into each hole: goat anti-mouse-HRP, 1:20,000. An incubator at 37℃for 1h.

(6) Taking out the ELISA plate, discarding the inner liquid, washing the plate for 4 times, adding 100 mu L of TMB color development liquid into each hole, and determining the color development time according to the color depth, wherein the color development time is generally 37 ℃ for 15min.

(7) The reaction was terminated by adding 100. Mu.L of 1M HCl solution to each well. Immediately, the wells with OD values greater than 2.1 times the OD value of the set negative control were titered as the sample by a 450nm reading on an microplate reader.

Analysis of results: the test results of the serum titers of the mice 1# and 4# and 6 post-immune mice show that the serum titer of the mice 1# can reach 1093.5K, the post-immune serum titer of the mice 6 is obviously improved, cell fusion experiments can be arranged, and the positive rate of fusion cells is based on the actual results; the immune serum titer of the 4# mouse 6 after the immune is about 40.5K, the immune serum titer is not obviously improved through two boosting experiments, and the 4# mouse is not subjected to the boosting experiment.

Step 2:

(1) Culturing trophoblast:

BALB/c mouse peritoneal macrophages were used as feeder cells. 1-2 days before fusion, BALB/c mice were euthanized by cervical vertebra blanching, and after sterilization and fixation of 75% alcohol body surfaces, abdominal skin was cut off with sterile scissors to expose the peritoneum. 5-10mL DMEM basal medium (without serum) is injected into the mice abdominal cavity with a syringe, taking care that the syringe needle does not pierce the intestinal tract and viscera in the abdominal cavity, so as not to contaminate the abdominal macrophages. Repeatedly blowing and flushing for several times, recovering abdominal cavity flushing liquid, centrifuging at 1000rpm for 10min, collecting, and discarding supernatant. The cells were resuspended in DMEM containing 20% fetal bovine serum to adjust the cell concentration to 1X 10 ⁵ CFU/mL. The cell suspension was added to a 96-well plate at 0.1 mL/well, 37℃and 5% CO ₂ The incubator was cultured overnight.

(2) Preparation of myeloma cells:

one week prior to fusion, SP2/0 cells were grown expanded in DMEM medium containing 10% FBS. By the time of fusion, the cells grew to approximately 6T 25 cell flasks, and SP2/0 cells were collected on the day of fusion into 50mL centrifuge tubes, 1500rpm, and centrifuged for 5min. The supernatant was discarded, then 20mL of DMEM basal medium was added, the cells were blown off and counted.

(3) Spleen cell preparation:

six immunized mice with serum ELISA titers meeting the requirements were immunized 3 days before fusion, 100 μg of PK34 antigen was injected intraperitoneally, freund's adjuvant was not included, emulsification was not performed, and intraperitoneal injection was directly performed. Mice to be fused were euthanized on the day of fusion by cervical dislocation. Soaking in 75% alcohol for 5min. Spleens were aseptically removed and placed in petri dishes with 10mL DMEM basal culture. The screen was placed in another plate, the spleen was transferred to the screen and the spleen was ground with the syringe core. DMEM was added to the screen and the screen was rinsed to allow more spleen cells to collect in the dish. The cells were transferred to a 50mL centrifuge tube, and the spleen cells were washed once with serum-free DMEM and centrifuged at 1500rpm for 5min. The spleen cells were collected and removed with a red blood cell lysate, washed once with serum-free DMEM, and centrifuged at 1500rpm for 5min. Spleen cell counts were collected.

(4) Cell fusion:

myeloma cells and spleen cells were mixed such that the myeloma cell to spleen cell number ratio was 1:2 is preferable. The cells were placed in 50mL centrifuge tubes, diluted with DMEM basal medium, centrifuged at 1500rpm for 5min, and the supernatant discarded. The electrofusion solution was added to the centrifuge tube, the tube was shaken to homogenize the cells, and then centrifuged at 1500rpm for 5min, the supernatant was discarded, and the cells were washed once. Then adding electrofusion solution to adjust the cell density to 2×10 ⁷ CFU/mL, then 2mL of cell suspension was added to the fusion pool for electrofusion on-board experiments. The ac calibration voltage is set to about 35V, the ac duration is within 60s, the low voltage mode (LV mode) dc integration voltage is set to about 260V, and the dc pulse width is set to about 40 μs. After electrofusion, standing for 10min, centrifuging the fused cells at 1000rpm for 5min, collecting the discarded supernatant, re-suspending the fused cells with HAT DMEM medium, and adding the fused cells into a 96-well cell culture plate containing trophoblast prepared 1-2 days in advance, wherein the concentration of the fused cells is 100 mu L/well. The cell culture plates were then placed at 37℃in 5% CO ₂ Culturing in a cell culture box. The hybridoma cell cloning efficiency is over 50 percent, a small amount of cell fragments are contained, and the cell growth state is good after 6 days of fusion. HAT DMEM medium was completely changed 7 days after fusion. After 10 days of fusion, a first screening test of the supernatant of the fused cells is started, 100 mu L/hole of the supernatant is taken out, and 100 mu L/hole of new HAT DMEM culture medium is added, and a second screening test of the fused cells is performed in the same way as the first test.

(5) Fusion screening:

on the day before detection, 1. Mu.g/mL PK34 antigen was coated on ELISA plates (100. Mu.L/well) with phosphate buffer (PBS, pH 7.4) at 4℃overnight. The ELISA detection is carried out by sucking 100 mu L/hole of cell supernatant in the next day, and positive holes are judged according to ELISA results (positive holes are judged when the OD value of a sample hole/the OD value of a negative hole is more than or equal to 2.1). And (3) picking the positive holes detected by the whole plate by using a single-channel pipette, and performing a second confirmation detection to further confirm the positive holes. After cell fusion, 8 96-well cell culture plates were spread together, and 10 fused cell lines (1 A1, 1B5, 1F6, 1G4, 2B6, 3H11, 4G2, 4G7, 5B8, 6E 2) specific for PK34 were obtained by ELISA screening. Then, subcloning was performed on the positive well cells after the determination.

Step 3:

(1) Cell subcloning:

blowing cells in the positive holes, counting, adding N/4mL of DMEM culture medium (N is a cell counting result) into a centrifuge tube, taking 100 mu L of cell suspension into the centrifuge tube, keeping 1mL after blowing evenly, adding DMEM to 4mL, blowing evenly, and keeping 100 mu L (about 2 drops) at the bottom of the tube. Adding DMEM to 5mL into a centrifuge tube, uniformly mixing, dropwise adding into the first three rows of a 96-well plate, keeping about 1.8-2mL at the bottom of one drip tube in each hole, adding DMEM to 5mL, uniformly blowing, dropwise adding into D, E, F three rows of the 96-well plate, keeping about 1.5-1.8mL at the bottom of one drip tube, adding DMEM to about 2.8-3mL, uniformly blowing, dropwise adding into G, H rows of the 96-well plate, dropwise adding into one drip in each hole, observing under a microscope after 7-10 days, detecting a hole with clone growth, marking a monoclonal hole, picking positive monoclonal cells as far as possible for subcloning again, and picking out the monoclonal hole for enlarging culture strain after 100% of positivity is detected.

(2) Subcloning screening:

on the day before detection, 1. Mu.g/mL PK34 antigen was coated on ELISA plates (100. Mu.L/well) with phosphate buffer (PBS, pH 7.4) at 4℃overnight. The ELISA detection is carried out by sucking 100 mu L/well of cell supernatant in the next day, and positive wells are judged according to ELISA results (positive wells are judged when the OD value of a sample well/the OD value of a negative well is more than or equal to 2.1). And (3) picking the positive holes detected by the whole plate by using a single-channel pipette, and performing a second confirmation detection to further confirm the positive holes.

Positive clones were screened by ELISA as follows:

through ELISA screening detection, 4 monoclonal cell lines (1F 6C8, 2B6H2, 6E2B6, 6E2F 11) with higher specificity to PK34 are obtained.

Step 4: expanded culture and cryopreservation of hybridoma cells

Cell culture conditions: DMEM (high sugar) +15% fbs (BI company), 37 ℃,5% co ₂ The culture is carried out,

and (3) selecting a strong positive monoclonal cell strain 6E2B6 with higher specificity for PK34 protein, performing amplification culture, centrifuging at 1000rpm for 5min, discarding the supernatant, adding 1mL of DMEM complete medium to resuspend cells, transferring the cells into a 15mL sterile sharp-bottomed centrifuge tube, adding 4mL of DMEM to blow the cells uniformly, centrifuging at 1000rpm for 5min again. The supernatant was discarded (the amount of cell sediment at the bottom was observed, if cell sediment with a thickness of 0.5mm could be directly observed, the cells could be directly transferred to a T25 square flask for culture, if the cell sediment was small, the cells could be transferred to 2-3 24 wells for culture, and the state of the cells was observed the next day) and the sediment was resuspended and transferred to a T25 flask for continuous culture. The next day the cell status is observed, if the cell amount is smaller or the cell status is worse, feeder cells can be added to the dish or the serum concentration in the medium can be slightly increased.

Cell passage: t25 flask is grown to bottom density of 80-90%, the two sides are gently tapped for several times, whether cells fall off or not is observed, and the expansion culture is carried out according to the proportion of 1 to 3.

Cell cryopreservation solution: 90% of fetal bovine serum and 10% of dimethyl sulfoxide,

after the hybridoma cell density reached 90%, directly discarding the supernatant, gently beating both sides of the flask for several times, collecting cell suspension, centrifuging at 1000rpm for 5min, discarding the supernatant, and re-suspending thallus with cell frozen stock solution at a final concentration of 1×10 ⁶ CFU/mL is added into a freezing tube with written cell names, the freezing tube is transferred into a program cooling box, a refrigerator at minus 80 ℃ is used for overnight, and the freezing tube is transferred into liquid nitrogen for long-term storage the next day.

The hybridoma cell line of the anti-PK 34 monoclonal antibody is preserved in China Center for Type Culture Collection (CCTCC) NO: C202226, and the preservation date is as follows: 2022, 01, 26, classification naming: a hybridoma cell line which is resistant to PK34 monoclonal antibodies.

Example 3:

a preparation and identification method of anti-PK 34 monoclonal antibody,

step one: cell expression:

the monoclonal cell strain 6E2B6 is selected for expansion culture, and antibody expression is carried out.

Step two: antibody purification:

loading Protein G agarose gel medium into an affinity purification chromatographic column, slowly loading the supernatant of the 6E2B6 cells, eluting with glycine elution buffer after the antibody is combined to obtain the required purified antibody, immediately dialyzing overnight at 4 ℃ in phosphate buffer (PBS, pH 7.4), detecting the purity of the antibody by SDS-PAGE every other day, and measuring the concentration of the obtained antibody by using a BCA Protein concentration measuring kit. Finally, the concentration of the obtained antibody was 0.20mg/mL, and the volume was 6mL. The purified antibodies were subjected to SDS-PAGE and stained with Coomassie blue. The results are shown in FIG. 1, purified antibodies analyzed by SDS-PAGE, lane M: protein molecular weight standard; lane 1: antibody purification analysis results.

Step three: identification of antibodies:

(1) Titers of purified antibodies to PK34 were detected by ELISA techniques:

on the day before detection, 1. Mu.g/mL PK34 antigen was coated on ELISA plates (100. Mu.L/well) with phosphate buffer (PBS, pH 7.4) at 4℃overnight. After dilution of the purified antibody by a multiple ratio the next day (initial dilution 1:500), ELISA was performed to determine the potency of PK 34.

TABLE 1 purification of antibody Indirect ELISA detection results

The highest dilution of the antibody titer, namely the sample OD/blank OD, is more than or equal to 2.1, and the titer of the 6E2B6 purified antibody against the PK34 polypeptide reaches about 256K according to ELISA titer detection results.

(2) Antibody specificity identification:

the specificity of the purified antibodies was detected by Western blot technique. The PK34 protein is added into a polyacrylamide gel sample adding hole, 100V is run to the junction of the concentrated gel and the separation gel, and then the voltage is increased to 160V until the electrophoresis is finished. And after electrophoresis, taking down the gel to perform wet transfer, and performing constant-current 280mA transfer for about 1h. After the electrotransfer, the membrane is taken off, washed with PBST for 4 times each for 5min, and then is put into 5% skim milk powder sealing liquid for sealing for 1h at 37 ℃. The purified antibodies were diluted 1:200 with PBST and the membranes were incubated overnight at 4℃in the purified antibody dilutions. The next day after membrane removal, the membrane was washed 4 times with PBST for 5min each, and the secondary antibody was diluted with a blocking solution containing 5% skimmed milk powder at a ratio of 1:20000. After incubating the membranes in secondary anti-dilution for 1h at 37 ℃, the membranes were removed and placed in a clean box for 4 membrane washes of 5min each time. Development using ECL luminophore, exposure, results are shown in fig. 2, lane M: protein molecular weight standard; lane 1 antibody-PK 34 WB assay results.

Example 4:

full-length sequencing of the genes of anti-PK 34 monoclonal antibodies:

the method comprises the following steps:

step 1: total RNA is extracted by hybridoma cell lysis:

taking a proper amount of hybridoma cells of the PK34 monoclonal antibody, and extracting total RNA of the hybridoma cells after cell lysis.

Step 2: reverse transcription of RNA into cDNA is carried out by using RACE technology, and then the full length of heavy chain and light chain is obtained by PCR amplification by taking cDNA as a template:

by RACE technology (cDNA end rapid amplification technology), specific primers are designed according to known sequences, 3'RACE is utilized to obtain 3' end sequences (gene specific primers→3 'ends), 5' RACE is utilized to obtain 5 'end sequences (gene specific primers→5' ends), and finally complete cDNA sequences are obtained. And then, using the upstream and downstream primers of the 5'RACE and the 3' RACE to obtain gene amplification products of the heavy chain and the light chain of the monoclonal antibody through PCR amplification, and using ddH2O as a template to complete the operation of the whole system so as to set a negative control group of PCR amplification.

Step 3: ligating the target fragment to the vector using a ligase and transforming the ligation product into E.coli competent cells, and then selecting a monoclonal for screening sequencing:

the above PCR product was purified and recovered using a PCR product recovery kit (Vazyme Co.), and the recovered product and the ligation vector pUC57 vector were subjected to single cleavage using EcoR V restriction enzyme. The purified product after cleavage and the carrier were then reacted at 25℃for 30 minutes using T4 ligase (Thermo Co.) to prepare a ligation reaction system using the following Table 2.

TABLE 2 ligation reaction System

TABLE 3 colony System

The recombinant vector was transformed into TOP10 competent cells, and 700. Mu.L of sterile LB medium without antibiotics was added thereto, and resuscitated at 37℃for 60 minutes at 200rpm after mixing. The recovered competent cells were plated on ampicillin-resistant LB plates and cultured at 37 ℃. After about 10 hours, monoclonal colonies were visible. Colony PCR was used to verify after picking the monoclonal colonies, the specific bacterial detection system is shown in Table 3, and the colony PCR program settings are shown in Table 4. Monoclonal colonies identified by PCR as positive were subjected to extensive culture. Plasmid miniprep (Axygen) was performed on the colonies after the expansion culture, and the plasmids were subjected to nucleotide sequencing based on Sanger sequencing.

TABLE 4 colony PCR program settings

Step 4: sequencing result analysis and annotation:

and (3) comparing the nucleotide sequence and the amino acid sequence of heavy and light chain sequencing results of the anti-PK 34 monoclonal antibody in NCBI and IMGT databases respectively, and realizing homology analysis. The resulting sequences were compared for homology with other antibody genes previously reported and analyzed for germ line gene origin. And the leader sequence (signal peptide) of the heavy and light chain of the antibody, the sequences of the FRs and CDRs of the variable region, and the sequences of the constant region of the antibody were determined by NCBI, FR-IMGT and CDR-IMGT analyses. Experimental results: nucleotide sequence alignment shows that the heavy chain variable region gene sequence of the anti-PK 34 monoclonal antibody has the same sequence as that of GenBank: the mouse Ig heavy chain variable region gene sequence of MH765571.1 has the highest homology of 278/292 (95%). The light chain variable region gene sequence of the anti-PK 34 monoclonal antibody is numbered GenBank: the gene sequence homology of the mouse Ig light chain variable region of NG_005612.1 is highest and is 298/305 (98%). The amino acid sequence alignment shows that the heavy chain variable region amino acid sequence of the anti-PK 34 monoclonal antibody has the same sequence as that of the heavy chain variable region with the serial number of GenBank: the murine Ig heavy chain variable region gene sequence of AEX91931.1 has the highest homology of 98/118 (83%). The light chain variable region gene sequence of the anti-PK 34 monoclonal antibody is numbered GenBank: the mouse Ig light chain variable region gene sequence of ADO17787.1 has the highest homology of 107/112 (96%). The homology analysis shows that the nucleotide sequence of the heavy and light chain variable region of the anti-PK 34 monoclonal antibody is derived from a mouse germ line gene, but is not completely consistent with the gene sequences of various monoclonal antibodies reported in the prior art, which shows that the invention has uniqueness on the gene sequence. The amino acid sequence of the heavy and light chain variable region of the anti-PK 34 monoclonal antibody is a murine protein, and although the amino acid sequence has homology with other protein amino acid sequences, the amino acid sequence which is completely the same as the amino acid sequence of the invention is not found, so that the amino acid sequence of the invention is unique.

The 3 CDR sequences of the heavy chain variable region of the anti-PK 34 monoclonal antibody are shown in SEQ ID NO.3 part, and specifically:

CDR1：Gly Phe Thr Phe Thr ASn Tyr Gly

CDR2：Ile Asn Thr Asp Thr Gly Glu Pro

CDR3：Ala Lys GIu Asp Asp Ser Gly Ala Ile Asp Tyr

the 3 CDR sequences of the light chain variable region of the anti-PK 34 monoclonal antibody are shown in SEQ ID NO.4, and specifically:

CDR1：Gln Ser Ile Val His Asn Asn Gly Asn Thr Tyr

CDR2：Lys Val Ser

CDR3：Phe Gln Gly Ser His Phe Pro Pro Thr

the above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the essence of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Weifang medical college

<120> anti-PK 34 monoclonal antibodies and hybridoma cell lines, and methods for preparing and purifying same

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 354

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcacgatc 60

tcctgcaaga cttctggctt taccttcaca aactatggaa tgatctgggt gaggcagcct 120

ccaggaaagg gtttcaagtg gatgggctgg ataaacaccg acactggaga gccaacatat 180

gctgatgagt tcaagggacg gtttgccttc tctttggaga cctctgccag cactgcctat 240

ttgcagatca gcaacctcaa aaatgaagac atggcttcat atttctgtgc aaaagaggat 300

gattccgggg ctatagacta ctggggtcaa ggaacctcag tcaccgtctc ctca 354

<210> 2

<211> 336

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

gatgttttga tgacccaaac tcccctctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gttctagtca gagcattgta cataataatg ggaacaccta tttagaatgg 120

tacctgcaga aaccaggcca gtctccaagg ctcctgatct acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggcagt ggttcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tattactgct ttcaaggttc acattttcct 300

cccacgttcg gctcggggac aaagttggaa ataaaa 336

<210> 3

<211> 118

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> V-region

（26）…（33）

Heavy chain CDR1 region

V-region

（51）…（58）

Heavy chain CDR2 region

V-region

（97）…（107）

Heavy chain CDR3 region

<400> 3

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Thr Ile Ser Cys Lys Thr Ser Gly Phe Thr Phe Thr Asn Tyr

20 25 30

Gly Met Ile Trp Val Arg Gln Pro Pro Gly Lys Gly Phe Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asp Thr Gly Glu Pro Thr Tyr Ala Asp Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Met Ala Ser Tyr Phe Cys

85 90 95

Ala Lys Glu Asp Asp Ser Gly Ala Ile Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> V-region

（27）…（37）

Light chain CDR1 region

V-region

（55）…（57）

Light chain CDR2 region

V-region

（94）…（102）

Light chain CDR3 region

<400> 4

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Ser Ser Ser Gln Ser Ile Val His Asn

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Phe Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 5

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atggattggc tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagca 57

<210> 6

<211> 57

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc caacagt 57

<210> 7

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Met Asp Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ala Gln Ala

<210> 8

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Met Lys Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala

1 5 10 15

Ser Asn Ser

<210> 9

<211> 975

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gccaaaacga cacccccatc tgtctatcca ctggcccctg gatctgctgc ccaaactaac 60

tccatggtga ccctgggatg cctggtcaag ggctatttcc ctgagccagt gacagtgacc 120

tggaactctg gatccctgtc cagcggtgtg cacaccttcc cagctgtcct gcagtctgac 180

ctctacactc tgagcagctc agtgactgtc ccctccagca cctggcccag cgagaccgtc 240

acctgcaacg ttgcccaccc ggccagcagc accaaggtgg acaagaaaat tgtgcccagg 300

gattgtggtt gtaagccttg catatgtaca gtcccagaag tatcatctgt cttcatcttc 360

cccccaaagc ccaaggatgt gctcaccatt actctgactc ctaaggtcac gtgtgttgtg 420

gtagacatca gcaaggatga tcccgaggtc cagttcagct ggtttgtaga tgatgtggag 480

gtgcacacag ctcagacgca accccgggag gagcagttca acagcacttt ccgctcagtc 540

agtgaacttc ccatcatgca ccaggactgg ctcaatggca aggagttcaa atgcagggtc 600

aacagtgcag ctttccctgc ccccatcgag aaaaccatct ccaaaaccaa aggcagaccg 660

aaggctccac aggtgtacac cattccacct cccaaggagc agatggccaa ggataaagtc 720

agtctgacct gcatgataac agacttcttc cctgaagaca ttactgtgga gtggcagtgg 780

aatgggcagc cagcggagaa ctacaagaac actcagccca tcatggacac agatggctct 840

tacttcgtct acagcaagct caatgtgcag aagaacaact ggggggcagg aaatactttc 900

acctgctttg tgttccatga gggcctgcac aacccccata ctgagaagag cctctcccac 960

tctcctggta aatga 975

<210> 10

<211> 324

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cgggctgatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60

ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120

tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180

agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240

cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300

agcttcaaca ggaatgagtg ttga 324

<210> 11

<211> 324

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Asn Asn Trp Gly Ala Gly Asn Thr Phe Thr Cys Phe Val

290 295 300

Phe His Glu Gly Leu His Asn Pro His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 12

<211> 107

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

Claims (7)

1. A hybridoma cell line against a PK34 monoclonal antibody, characterized by: the preservation number of the hybridoma cell line is CCTCC NO: C202226.

2. The hybridoma cell line against the PK34 monoclonal antibody according to claim 1, characterized in that: the hybridoma cell line secretes an anti-PK 34 monoclonal antibody.

3. A monoclonal antibody against PK34 secreted by the hybridoma cell line according to any one of claims 1-2, characterized in that: the heavy chain variable region gene sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region gene sequence is shown as SEQ ID NO. 2.

4. A monoclonal antibody according to claim 3, characterized in that: the polypeptide sequence of the heavy chain variable region gene is shown as SEQ ID NO.3, and the polypeptide sequence of the light chain variable region gene is shown as SEQ ID NO. 4.

5. A monoclonal antibody according to claim 3, characterized in that: the heavy chain signal peptide gene sequence of the monoclonal antibody is shown as SEQ ID NO.5, and the light chain signal peptide gene sequence is shown as SEQ ID NO. 6;

the polypeptide sequence coded by the heavy chain signal peptide gene is shown as SEQ ID NO.7, and the polypeptide sequence coded by the light chain signal peptide gene is shown as SEQ ID NO. 8.

6. A monoclonal antibody according to claim 3, characterized in that: the heavy chain constant region gene sequence of the monoclonal antibody is shown as SEQ ID NO.9, and the light chain constant region gene sequence is shown as SEQ ID NO. 10;

the polypeptide sequence of the heavy chain constant region gene is shown as SEQ ID NO.11, and the polypeptide sequence of the light chain constant region gene is shown as SEQ ID NO. 12.

7. A method of purifying an anti-PK 34 monoclonal antibody according to any one of claims 3 to 6, wherein:

step 1: subjecting the hybridoma cell of claim 1 or 2 to antibody expression;

step 2: slowly loading the cell supernatant by using an affinity purification chromatographic column, and eluting with glycine elution buffer after the antibody is combined to obtain the required purified antibody;

step 3: the antibodies were dialyzed overnight at 4℃in PBS, and purity and concentration were measured every other day.

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