CN112062846A

CN112062846A - G-17 monoclonal antibody and application thereof

Info

Publication number: CN112062846A
Application number: CN202010343881.2A
Authority: CN
Inventors: 李因来; 徐东鑫; 何燕燕
Original assignee: Hangzhou Biogenome Biotechnology Co ltd
Current assignee: Hangzhou Biogenome Biotechnology Co ltd
Priority date: 2020-04-27
Filing date: 2020-04-27
Publication date: 2020-12-11

Abstract

The invention discloses a G-17 monoclonal antibody which is an IgG type and is prepared from monoclonal cell strains GC-1 and GN-1, wherein the cell strains GC-1 are preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation date of 2019, 1 month and 16 days and the preservation number of CGMCC No. 17088; the cell strain GN-1 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17089. The monoclonal antibody comprises C100 and N100, an antibody N100 is used as a coating antibody, C100 is used as a labeled antibody, a DAS-ELISA method is established, and the content of G-17 in natural serum is detected. The invention has the beneficial effects that: the monoclonal antibody can be specifically combined with G-17, and has high recognition capability and efficiency. The monoclonal antibody can be effectively applied to medicine preparation, and the targeting property and specificity of the medicine are increased.

Description

G-17 monoclonal antibody and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a G-17 monoclonal antibody and application thereof.

Background

Gastrin is an important gastrointestinal peptide hormone, first discovered and named by Edkins, a british scholar in 1905. Gastrin is synthesized and secreted by G cells located in the mucosa proximal to the antrum and duodenum. The synthesis of gastrin progresses from progastrin and glycine-extended gastrin to mature gastrin, the intermediate product of the synthesis of the former two gastrins is unamidated gastrin, the latter gastrin is amidated gastrin, and more than 95% of active gastrin human body is α -amide gastrin. Amidated gastrins include G17, G34, G14, G6, G52, G71, where G17 is the predominant form of gastrin in the antrum, the predominant form of gastrin in the blood after meals, and G34 is the predominant form of gastrin the duodenum, the predominant form of gastrin between meals. Gastrin exerts biological effects through a series of signal transduction upon binding to the gastrin receptor. Gastrin has the same carboxy-terminal pentapeptide sequence as cholecystokinin, and therefore its receptor is identical to cholecystokinin receptor (CCKR), and is also known as CCKR. CCKR has two main types of CCK-A and CCK-B receptors, and gastrin exerts a biological effect mainly by binding to the CCK-B receptor.

In Chronic Atrophic Gastritis (CAG), serum G17 has a correlation with the location and extent of atrophy of gastric mucosa. Cao et al found in a case-control study that serum G17 was lower in the antral atrophy group than in the control group, serum G17 was higher in the gastric atrophy group than in the control group, and serum G17 was lower but higher in the multifocal atrophy group than in the antral group. When the analysis result is that the gastric antrum is atrophied, the number of G cells is less, and the synthesis of G17 is reduced; when the stomach body is atrophied, the number of acid secretion cells is reduced, the stomach is in a low acid state, and the gastrin is increased due to negative feedback of the gastric acid axis of the gastrin. Compared with a normal control group, the study of the exemplar and the like respectively compares the gastric mucosa light, medium and severe atrophy groups, the content of serum G17 is obviously increased when the gastric mucosa is slightly atrophic, no obvious difference exists when the gastric mucosa is moderately atrophic, and the content is obviously reduced when the gastric mucosa is severely atrophic; compared with the normal control group, the number of G cells has no significant difference when the gastric mucosa is slightly atrophic. And in severe atrophy in the gastric mucosa, is significantly reduced.

The Chenmoryer et al study found that serum G17 levels were significantly elevated in all patients in the gastric ulcer group compared to the non-atrophic gastritis group (P < 0.05). Zhangzhong et al found that the level of serum G17 was progressively increased from the normal case through superficial gastritis to gastric erosion or ulcer by examining the G17 level in 3906 serum samples. Elevated serum G17 levels may be associated with an inflammatory response of the gastric mucosa and with h.

The level of serum G17 in gastric cancer patients is related to lesion sites, clinical stages and pathological tissue typing. Among 164 patients with gastric cancer, the patients were classified into antral carcinoma group, corpus carcinoma group, fundus and cardia carcinoma group, and serum total gastrin level was detected, and the result showed that the serum gastrin level of fundus and cardia carcinoma was much higher than that of corpus carcinoma, antrum carcinoma and normal control group, and was higher with the clinical stage of gastric cancer; the gastrin level group of the gastric body cancer is higher than that of the control group, and the cancer group of the antrum is lower than that of the control group, but the difference between the two groups has no statistical significance. The obvious increase of serum G17 in fundus, cardia and corpus carcinoma is probably due to the fact that cancer tissues destroy acid secreting glands and vagus nerve endings in large quantity, so that gastric acid secretion is lost, and the feedback of serum gastrin level is increased. Konturek et al determined serum gastrin 72 gastric cancer groups and 77 control groups, and found that serum gastrin in the gastric cancer group is higher than that in the control group, and the number of patients with gastrinemia in the intestinal gastric cancer group is obviously greater than that in the diffuse type and mixed type gastric cancer groups. Gastrin has high affinity with CCK-2 receptor in upper gastrointestinal tract mucosa, and can activate multiple signal pathways such as cell proliferation, anti-apoptosis, inflammatory reaction and the like after being combined, induce acid secretion and promote tumorigenesis. Morton et al established a high gastrin animal model and found that the gastric mucosal cells undergo malignant transformation. Takaishi et al, when studying the relationship between gastric cancer and h.pylori and gastrin, suggested that h.pylori infection is a initiating factor of gastric cancer, and gastrin is an important factor for promoting the development of gastric cancer.

Disclosure of Invention

In order to detect the content of G-17 in natural serum, a G-17 monoclonal antibody and application thereof are provided. The invention is realized by the following technical scheme:

the invention provides a G-17 monoclonal antibody, which is an IgG type and is prepared from monoclonal cell strains GC-1 and GN-1, wherein the cell strain GC-1 is stored in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of No.1 Hosieboldo, North West Lu, of the Chaoyang area, Beijing, the storage date is 2019, 1 month and 16 days, the storage number is CGMCC No.17088, and the monoclonal antibody is named as a hybridoma cell secreting anti-G-17 monoclonal antibody in a classified manner; the cell strain GN-1 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, No. 3 of Xilu No.1 of Suzhou province of the rising area of Beijing city, the preservation date is 2019, 1 month and 16 days, the preservation number is CGMCC No.17089, and the cell strain is classified and named as a hybridoma cell secreting anti-G-17 monoclonal antibody.

Preferably, the monoclonal antibody comprises C100 and N100, wherein C100 is prepared from GC-1 and N100 is prepared from GN-1.

The invention also provides application of the G-17 monoclonal antibody, which utilizes the antibody N100 as a coating antibody and C100 as a marker antibody to establish a DAS-ELISA method and detect the G-17 content in natural serum.

Preferably, the amino acid sequence of C100 is:

Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2。

preferably, the amino acid sequence of N100 is: Pro-Glu-Gly-Pro-Trp-Leu-Glu-Glu.

Preferably, the classification of C100 is IgG2 a.

Preferably, the typing of N100 is IgG 1.

Compared with the prior art, the invention has the beneficial effects that:

(1) the monoclonal antibody can be specifically combined with G-17, and has high recognition capability and high efficiency.

(2) The monoclonal antibody can be effectively applied to medicine preparation, and increases the targeting property and specificity of the medicine.

Detailed Description

The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the present invention is not limited to the following examples, and that various changes or modifications may be made without departing from the scope of the invention; and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1:

first, obtaining hybridoma cell and preparing monoclonal antibody thereof

1. Animal immunization

The immunogen is a recombinant antigen purchased from Jinsbane technology (Nanjing) Inc., under the trade designations PE2594 (GN) and PE2595 (GC). The amino acid sequence of GN is:

Pro-Glu-Gly-Pro-Trp-Leu-Glu-Glu; the amino acid sequence of GC is:

Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2. BALB/c female mice, 18-20g in body weight, 6 weeks old were immunized with the recombinant protein. Mixing 100 mu G/G of G-17 recombinant protein with Freund's complete adjuvant, emulsifying completely, injecting 0.3 mL/G subcutaneously via dorsoventral region, emulsifying 80 mu G/G of antigen and Freund's incomplete adjuvant completely every 2 weeks, injecting 0.3 mL/G intraperitoneally, immunizing for 4 weeks, collecting blood from mouse eyeball, detecting serum titer, and collecting titer 1:10⁵And the spleen strengthening immunization is carried out on the above mice, when the spleen is immunized, the amount of the recombinant antigen is 30 mu g/mouse, and spleen cells are taken 3 days later for cell fusion.

2. Preparation of feeder layer cells

Taking BALB/c mouse abdominal cavity macrophages and thymocytes as feeder cells, and the specific operation method is as follows: BALB/c mice were bled and killed, the body surface was soaked in 75% ethanol for sterilization, and then the abdominal skin was cut from the back abdomen with sterilized scissors and forceps to expose the peritoneum. 4ml of IMDM medium was added to the abdominal cavity by syringe, and the abdomen was gently squeezed with fingers and repeatedly washed to recover the washing solution. The thymus of the mouse was removed, crushed, washed with IMDM medium, and the wash solution was recovered. And mixing the two recovered solutions, centrifuging at 1200rpm for 3min, and removing the supernatant to obtain feeder layer cells.

3. Cell fusion

Mixing immune mouse spleen cell and mouse myeloma cell (SP2/0) at ratio of 7:1 in serum-free IMDM culture medium, centrifuging at 1300rpm for 3min, removing culture medium, adding 1mL 50% PEG (molecular weight 1500) in 37 deg.C water bath, fusing for 2min, terminating fusion with serum-free IMDM culture medium, centrifuging at 1300rpm for 3min, suspending precipitate with HAT culture medium, and adding feeder layer cellThe cells were split into 96-well feeder cells-containing cell plates and placed at 37 ℃ with 5% CO₂The cell culture box.

4. Screening of hybridoma cells and cloning thereof

After culturing for 5 days in a cell culture box, when fused cells cover 10-30% of the bottom of a hole, screening positive holes secreting antibodies by using a conventional indirect ELISA method, namely using G-17 recombinant protein as an antigen, diluting the recombinant protein with CB to 0.5 mu G/ml to coat a 96-hole enzyme label plate, coating the enzyme label plate at 50 mu l/hole and at 4 ℃ overnight, after patting dry, sealing the enzyme label plate with 1% BSA-containing PBS buffer solution (200 mu l/hole), sealing the enzyme label plate at 37 ℃ for 2 hours, and patting dry the enzyme label plate for later use; adding 50 μ l/well of cell culture supernatant to be detected into the ELISA plate, reacting at 37 deg.C for 30min, washing, draining, adding 50 μ l/well HRP-labeled goat anti-mouse IgG, incubating at 37 deg.C for 30min, washing, draining, adding 50 μ l/well TMB developing solution, developing at 37 deg.C in dark for 10min, adding 25 μ l/well 2M H₂SO₄The reaction was terminated and the value read at 0D 450. Determination of positive wells: OD450 value/negative control value is more than or equal to 2: 1. And selecting the cell wells with strong positive reaction, and cloning by a limiting dilution method. After three to four times of cloning screening, the positive rate of the monoclonal cell strain is 100 percent, and the monoclonal cell strain is determined to be a stable cell strain. After the enlarged culture, the culture medium is used for ascites preparation and liquid nitrogen preservation. The two monoclonal cell lines were designated GC-1 and GN-1, respectively.

5. Preparation and purification of monoclonal antibody ascites

Taking F1 mouse of about 8 weeks old, injecting 0.3-0.5mL paraffin into abdominal cavity, injecting 8 × 10 into abdominal cavity 7-10 days later⁵The hybridoma cells can be injected for 7-10 days to show that the abdomen of the mouse is obviously enlarged, ascites is collected by an injection needle, the ascites is centrifuged for 3min at 8000rpm, and the supernatant is collected to be the monoclonal antibody ascites. Diluting 1 volume of ascites with 2 volumes of 0.06M acetic acid buffer solution with pH 4.8, adding caprylic acid (30 mu L/mL ascites) at room temperature while stirring, clarifying at 4 deg.C for 1h, centrifuging at 12000rpm for 20min, collecting supernatant, precipitating immunoglobulin with 50% saturated ammonium sulfate, standing at 4 deg.C for 2h, centrifuging at 3000rpm for 20min, dissolving the precipitate with 2 times volume of PBS solution, and performing flow dialysis at 4 deg.C for 24h to obtain purified ascites antibody, and storing at-70 deg.C.

Second, screening preparation of paired antibodies

1. HRP labeling of antibodies

Dissolving 1mg HRP in 0.5ml distilled water, and adding 0.2ml NaIO₄The solution (0.06mol/L) is mixed evenly and placed under the condition of light shielding at 4 ℃ for 30 min. 0.2ml of ethylene glycol solution (0.16mol/L) was added thereto, and the mixture was stirred gently at room temperature (about 20 ℃ C.) for 30min in the absence of light. Adding 1mg antibody solution, mixing, placing into dialysis bag, and dialyzing in CBS solution under dark condition for 6 hr (or overnight at 4 deg.C). The reaction solution was removed from the dialysis bag and 50. mu.l of NaBH was added₄The solution (5mg/ml) was mixed well and placed at 4 ℃ under light-shielding conditions for 2 h. Slowly adding equal volume (about 1.2ml) of saturated ammonium sulfate solution, mixing, placing at 4 deg.C in dark for 30min, centrifuging at 10000rpm for 10min to remove supernatant, re-dissolving with 200ul PBS for precipitation, and dialyzing in PBS for 12-18h, wherein the solution is changed once. Taking out the solution, measuring the volume, adding glycerol with the same volume, mixing well, and storing at-20 deg.C for use.

2. Screening for partner antibodies

The coated antibody was diluted to 4. mu.g/ml with CB, added to wells of an ELISA plate at 100. mu.l per well, and coated overnight at 4 ℃. The plate was blotted dry and 200. mu.l of blocking solution (1% BSA in PBS) was added to each well, blocked for 1-2h at 37 ℃ and blotted dry. Positive blood samples were diluted to 2ng/ml with PBS and loaded at 100. mu.l per well, with negative blood samples as controls. After incubation at 37 ℃ for 1h, the plates were washed 3 times in 0.05% PBST. The enzyme-labeled antibody was diluted to 1000-fold working solution with a 1% BSA-containing wash solution, 100. mu.l was added to each well, incubated at 37 ℃ for 1 hour, and then the plate was washed 4 times and patted dry. Mu.l of TMB developing solution was added to each well, and the mixture was incubated at 37 ℃ for 10 min. Add 50. mu.l of stop buffer to each well and read the OD on a microplate reader at 450 nm. The results are as follows:

thirdly, performance evaluation and related parameters of paired antibody C100/N100

1. Specific detection of paired antibodies

According to a specificity detection method, the specificity of C100/N100 pairing is detected, 25 parts of positive serum and 25 parts of negative serum are used together, P/N is more than or equal to 2 as positive, and the result is as follows:

2. typing and identification of monoclonal antibodies

The antibody typing identification kit SBACLONITyping System-HRP (Southern Biotech, cat:5300-05) was used for identification according to the instruction method.

The specific method comprises the following steps:

coating: diluting the capture antibody to a concentration of 5-10. mu.g/mL with Borate Buffer (BBS) at pH8.2 or Phosphate Buffer (PBS) at pH7.4, adding 100. mu.L per well;

covering the enzyme label plate with a cover or a preservative film, and incubating for at least 12 hours in a humid environment at the temperature of 2-8 ℃;

sealing: spin-drying, washing with PBS or BBS containing 0.05% Tween for 3 times; spin-drying, adding PBS or BBS containing 1% BSA, 200 μ L per well;

standing the antibody-coated plate at room temperature for at least 1h to block free binding sites on the plate;

adding 100 mul hybridoma supernatant or antibody to be detected into each hole, covering, and incubating for 1h at room temperature by mild oscillation or overnight at 2-8 ℃;

sixthly, washing for 3 times by using a washing liquid;

seventhly, diluting HRP-labeled detection antibody 1:250-1:500 with BBS/BSA, adding 100 μ L of conjugate into a plate, covering the plate, and shaking at room temperature for 1h or standing overnight at 2-8 ℃;

washing 5 times with the lotion;

ninthly, preparing ABST substrate stock solution, namely dissolving 15mgABST powder in 1mL double distilled water, storing at 2-8 ℃ in a dark place, and stabilizing within 4 weeks;

preparation of substrate solution for R (R) - -525mg citric acid to 50mL double distilled waterStirring until the mixture is completely dissolved; adjusting the pH value to 4.0 by NaOH; to 10mL of citrate buffer was added 0.2mLABST stock and 10. mu.L, 30% H₂O₂；

Add 100. mu.L substrate buffer per well;

the plate reader was used to read at 405 nm.

The results are as follows:

ELISA detection OD405 counts:

and (3) typing identification results:

Claims

1. the G-17 monoclonal antibody is characterized in that the monoclonal antibody is of an IgG type and is prepared from monoclonal cell strains GC-1 and GN-1, wherein the cell strains GC-1 are preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17088; the cell strain GN-1 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2019, 1 month and 16 days, and the preservation number is CGMCC No. 17089.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody comprises C100 and N100, wherein C100 is prepared from GC-1 and N100 is prepared from GN-1.

3. The application of the G-17 monoclonal antibody is characterized in that the monoclonal antibody utilizes an antibody N100 as a coating antibody and C100 as a marker antibody to establish a DAS-ELISA method and detect the G-17 content in natural serum.

4. The use of a G-17 monoclonal antibody as claimed in claim 3, wherein the amino acid sequence of C100 is: Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH 2.

5. The use of a G-17 monoclonal antibody as claimed in claim 3, wherein the amino acid sequence of N100 is: Pro-Glu-Gly-Pro-Trp-Leu-Glu-Glu.

6. The use of a G-17 monoclonal antibody as claimed in claim 3, wherein said typing of C100 is IgG2 a.

7. The use of a G-17 monoclonal antibody as claimed in claim 2, wherein the classification of N100 is IgG 1.