CN111393530A

CN111393530A - New humanized T L R2 extracellular segment monoclonal antibody and preparation method and application thereof

Info

Publication number: CN111393530A
Application number: CN202010238550.2A
Authority: CN
Inventors: 肖斌; 李林海; 孙朝晖
Original assignee: Southern Theater Command General Hospital of PLA
Current assignee: Southern Theater Command General Hospital of PLA
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-07-10

Abstract

The invention discloses a monoclonal antibody and a preparation method thereof, wherein the monoclonal antibody is an antibody with a gene sequence of 259-position 278 amino acid coded in a human T L R2 extracellular region as a target spot, and also discloses application of the monoclonal antibody in preparation of a vaccine for inhibiting brucella, the monoclonal antibody can specifically recognize T L R2, and further inhibits the infection of brucella vaccine strains on human macrophage THP-1 by closing a first gate of the host cells invaded by the brucella, so that the monoclonal antibody has important value and significance in scientific research for inhibiting the infection of the brucella vaccine strains, and has wide popularization prospect.

Description

New humanized T L R2 extracellular segment monoclonal antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel humanized T L R2 extracellular domain monoclonal antibody, a preparation method and application thereof, and in particular relates to a monoclonal antibody of a gene sequence of 259-position amino acid-containing fragment of a novel humanized T L R2 extracellular domain coding gene, a preparation method and application thereof.

Background

T L R2 is a cell membrane protein which can be polymerized with other T L R family members to form heterodimers, thereby recognizing conserved molecules from pathogenic microorganisms, and this recognition mode is called pathogen-associated molecular pattern (PAMPs). T L R2 can further activate downstream signal pathways after being activated by PAMPs, thereby regulating the immune response of the host to pathogenic microorganisms.T L R2 is thought to mediate the apoptosis of host cells under the stimulation of bacterial lipoproteins.

At present, relevant documents show that T L R2 participates in recognition and immune response reaction of a host to brucella, T L R2 and T L R4 are important factors for increasing cytokine expression mediated by brucella surface protein BCSP31, Th1 immune response and macrophage regulation, the infection of brucella is remarkably reduced after T L R2 gene of the host cell is knocked down, and conversely, the infection of brucella is remarkably enhanced after T L R2 is over-expressed, which indicates that T L R2 is an important target for invasion of the brucella into the host cell and mediation of the host immune response.

In view of the important role of a membrane receptor T L R L in identification and infection of pathogenic microorganisms (Brucella), T L R L is taken as a target in the prior art, a corresponding small molecule inhibitor is developed, and the inhibition effect of the T L R L small molecule inhibitor on Brucella infection is evaluated, for example, Pragnesh et al takes Toll/I L3-1 receptor resistance (TIR) domains in the intracellular region of T L R L as a target, and a novel T L R L small molecule inhibitor C L (formula I) is designed and synthesized by using a computer-aided drug design scheme, only the influence of C L on the expression of a host macrophage T L R L signal pathway is evaluated, the study is only capable of inhibiting the interaction between T L R L and MyD L and inhibiting a downstream MAPK pathway and inhibiting the immune response mediated by T L R, and the synthesis of a T L-P complex (CPT L-P-L) and the study on the competitive binding of a protein complex of TNF-T L-CT-L and a T L-P-L (CST L-P-K complex) and a CST L-P-L-K complex is studied.

Therefore, the prior inhibitor or antibody research and development with the T L R2 as the target point are mostly designed and researched with the structure that the T L R2 intracellular region is directly combined with the second messenger as the target point, and the main purpose of the inhibitor or inhibitor is to inhibit the intracellular T L R2 mediated signal channel so as to inhibit the cellular immune response.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.

The invention aims to provide a monoclonal antibody, which aims to solve the problems in the prior art and is prepared by taking a gene sequence of T L R2 extracellular region coding 259-278 amino acid as a target spot, wherein the antibody can realize the purpose of blocking an important channel for brucella to invade host cells.

It is still another object of the present invention to provide a method for preparing a monoclonal antibody, by which a purified monoclonal antibody can be obtained for use in inhibiting brucella infection.

The invention also aims to provide the application of the monoclonal antibody in preparing the vaccine for inhibiting the brucella, which is applied to inhibiting the infection of the brucella vaccine strain on human macrophage THP-1.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a monoclonal antibody, which takes a gene sequence of a human T L R2 extracellular region coding 259-278 amino acid as a target.

Preferably, the amino acid sequence at position 259-278 is VKITDES L FQVMK LL NQISG.

The invention also provides a preparation method of the monoclonal antibody, which comprises the following steps:

step 1, immunizing a mouse, separating mouse spleen cells, fusing the mouse spleen cells and mouse bone marrow cells, culturing, screening positive hybridoma cells by using an E L ISA method, carrying out expanded culture on the preliminarily screened positive hybridoma cells, cloning the expanded cultured positive hybridoma cells by using a limiting dilution method, and screening out a positive hybridoma cell strain with a high OD value;

step 2: identifying the subtype of the monoclonal antibody by using a mouse monoclonal antibody subtype identification test strip on the cell supernatant of the obtained positive hybridoma cell strain;

and step 3: inoculating the positive hybridoma cell strain identified as the monoclonal antibody subtype in the step 2 into the abdominal cavity of the mouse, and separating and purifying the monoclonal antibody in the ascites fluid of the mouse.

Preferably, the ratio of mouse spleen cells and mouse bone marrow cells in step 1 is 4: density of 1.

Preferably, the obtained positive hybridoma cells are cloned twice in succession by limiting dilution, each clone is cultured in HT selective medium, and E L ISA screening is performed 7 days after cloning until the positive rate of the monoclonal cells is 100%.

The invention also provides application of the monoclonal antibody in preparation of a vaccine for inhibiting brucella.

Preferably, the vaccine strain is applied to inhibiting the infection of the brucella vaccine strain on human macrophage THP-1.

The invention discloses the following technical effects:

the T L R2 disclosed by the invention is a cell surface membrane protein, is also a first gate of a Brucella infected host cell, is an important receptor of a plurality of Brucella surface structure proteins, and is a monoclonal antibody, wherein the monoclonal antibody takes an antibody taking a gene sequence of a T L R2 extracellular segment coding 259 amino acid as a target spot, can specifically recognize T L R2, and can inhibit the infection of a Brucella vaccine strain on human macrophage THP-1 by closing the first gate of the Brucella invading the host cell.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a test strip for subtype identification of a Raybio mouse;

FIG. 2 is a subtype determination of T L R2(259-278aa) monoclonal cell line;

FIG. 3 shows the intracellular survival of the Brucella vaccine strain M5-90 after infecting human macrophage THP-1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Preparation of monoclonal antibodies

1.1 immunization of mice

a. 3 basic immunizations and 1 boosting immunizations are adopted in the immunization program, 3 healthy female BA L B/c mice (637#, 640#, 639#) with the body weight of about 18g and the age of 6-8 weeks are selected, and after the mice are bred for 1 week in an adaptive manner, negative blood is collected as a control;

b. stirring and emulsifying the immunogen and an equal volume of Freund's complete adjuvant at the first immunization (50 mu g/mouse), injecting subcutaneously and multiply points on the back, and then stirring and emulsifying the immunogen and an equal volume of Freund's incomplete adjuvant at the last immunization conventionally;

c.3 times of immunization, generally mixing and emulsifying 50 mu g of antigen and TiterMax in equal amount, injecting at multiple points on the back, measuring the titer 7 days later, wherein the titer reaches 8 ten thousand or more, preparing for boosting, adding no adjuvant for boosting, the boosting dose is 75 mu g, collecting blood from the eyeball 3 days later, separating serum and storing, and measuring the titer of antiserum; from the measurement results of table 1, it can be seen that: three mice had reached a titer of 32 ten thousand, and No. 640 mice were selected for spleen fusion.

TABLE 1T L R2(259-278aa) mouse antiserum titer determination

1.2 cell fusion

a. When the cells are fused, the spleen cells and myeloma cells of No. 640 mice are mixed according to a ratio of 4:1, and are fused under the fusion promoting effect of polyethylene glycol (PEG with the molecular weight of 1450), the fused cells are cultured in HAT selective culture solution, positive hybridoma cells are screened out by an indirect E L ISA method after 10 days, and the preliminarily screened positive hybridoma cells are subjected to expanded culture, the specific operation process is as follows:

(1) spleen cells and myeloma cells were measured as 4: mixing at a ratio of 1, washing with preheated basic culture solution for 1 time, centrifuging at 1000r/min for 5min, and removing supernatant.

(2) Standing for 1min, sucking residual culture solution with a pipette, lightly flicking the bottom of the centrifuge tube with hand to disperse the cells, and placing the bottom of the centrifuge tube in distilled water with 37 deg.C sterilization.

(3) The tube was slowly rotated and 1m L PEG was added over 1 min.

(4) Sucking the cell suspension into the pipette for 30s, slowly blowing into the bottom of the centrifuge tube, and standing in sterilized distilled water at 37 deg.C for 1 min.

(5) The preheated 3m L basic DMEM culture solution is added within 3min from slow to fast, and the centrifuge tube is rotated while adding.

(6) Adding preheated incomplete culture solution 10m L to dilute PEG within 3min, and removing melting promoting effect.

(7) 10m L of pre-heated incomplete culture medium was added within 2 min.

(8) Standing at room temperature for 10 min.

(9) Centrifuging at 800r/min for 4min, and discarding the supernatant.

(10) The pellet was suspended in preheated HAT medium, the cell suspension was added to a 96-well plate containing cultured cells at 100. mu. L/well, and the cell culture plate was then placed at 37 ℃ in 5% CO₂Cultured in an incubator.

The 5 plates were tested 10 days later for positive wells, italicized are positive and negative serum controls, and based on the test results, 5A5, 3E12, 5A12, 4B3, 4H11, 2E4 were selected for subclone screening, and the mouse fusion plates of Table 2T L R2(259-278aa) were tested.

TABLE 2T L R2(259-278aa) mouse fusion plate assay

b.T L R2(259-278aa) mouse subclone plate assay:

the obtained positive hybridoma cells are subcloned twice by a limiting dilution method, each subclone is cultured by an HT selective medium, and E L ISA screening is carried out 7 days after the subcloning until the positive rate of the monoclonal cells is 100%.

The hybridoma cell strain is cloned by adopting a limiting dilution method, and the specific operation process is as follows:

(1) cultured cells were prepared at the first 1d or the day of clone selection and added to 96-well cell culture plates at 100. mu. L/well, 37 ℃ with 5% CO₂Culturing in an incubator for later use.

(2) Cells in the strong positive well were gently pipetted to suspend them in the culture medium, aspirated from 100. mu. L to 2m L of complete medium, and counted.

(3) Diluting the cells to about 50 cells/m L with HT, taking 4.8m L, adding to the first two rows of 96-well cell culture plates, adding the rest 2.4m L HT culture solution, adding to the 4 th row and the 5 th row of the 96-well cell culture plates, adding 2.4m L HT culture solution, and so on until the addition of one 96-well cell culture plate is completed.

(4) Placing 96-well culture plate at 37 deg.C and 5% CO₂The residual positive cells are directly added into a culture bottle with the thickness of 50m L for culture and frozen storage.

(5) And 4d, half of the HT culture medium is used.

(6) At 7d, supernatants from 100 μ L wells were assayed for antibody in cell supernatants using indirect E L ISA.

(7) Selecting a monoclonal and strong positive hole (the judgment standard of the positive hole is that the OD value is more than 2.1 times of the negative OD value, namely positive, and the strong positive is the hole with the highest OD value in the positive hole) to subclone again until the positive clone reaches 100 percent.

(8) Transferring the selected strong positive hole cells of the hybridoma to a 6-hole culture plate for amplification culture.

(9) The expanded hybridoma cells are expanded again in a 50m L culture flask, centrifuged at 1000r/min for 5min, the supernatant is collected and titer-measured by indirect E L ISA, and a part of the hybridoma cells are frozen.

As shown in Table 3, the result is the second subcloning of T L R2(259-278aa), the result is marked with positive serum and negative serum control in italics, the positive rate is over 90%, the cloning wells with higher positive OD are selected, and the expanded culture is selected (in order: 24-6-10 cm plates).

TABLE 3T L R2(259-278aa) mouse subclone plate assay

c. The subtype of the monoclonal antibody of the mouse is identified by using a test strip for identifying the subtype of the monoclonal antibody, and the mouse subtype is determined by taking 20 mu L of the supernatant of the monoclonal cell strain according to the specification of the test strip.

FIG. 1 shows a mouse subtype identification test strip (Gold Series) of Raybio mouse, and the mouse subtype of the monoclonal cell line prepared according to the control of FIG. 1 is shown in FIG. 2, T L R2(259-278aa) -5A5 (L ambed, IgG 1).

1.3 preparation and purification of ascites antibodies

a. Selecting 8-12 week old female healthy BA L B/c mice, injecting paraffin oil into the abdominal cavity, wherein each mouse is 0.5m L, and injecting 1 × 106-5 × 106 monoclonal hybridoma into the abdominal cavity of each mouse 7-10 days later.

b. Centrifuging ascites at 10000r/min for 15min, removing cell components and other precipitates, fat, oil layer, etc., collecting the middle layer, and measuring antibody titer.

c. And (3) saturated ammonium sulfate precipitation, namely sucking 5m L of treated ascites, transferring the ascites into a small beaker, dropwise adding PBS 5.0m L passing through a 0.22 mu m filter membrane under the stirring condition, uniformly mixing, then dropwise adding 10m L of saturated ammonium sulfate solution (pH7.4), continuously and slowly stirring for 30min, standing for 2h, centrifuging at 10000r/min for 15min, discarding supernatant, re-suspending the precipitate with PBS passing through the 0.22 mu m filter membrane, and then passing the re-suspended solution through the 0.22 mu m filter membrane.

d. Selecting different purifying columns of GE Healthcare company according to different subtypes of the antibody, wherein the columns of IgG and IgM all have corresponding instructions, and preparing different buffers according to the instructions to purify, taking IgG antibody purification as an example, firstly balancing the columns by using a binding buffer solution, an elution buffer solution and a regeneration buffer solution, generally balancing 5 column volumes, then loading the resuspended ascites buffer solution at the speed of 1m L/min, balancing by using the binding buffer solution after loading, then washing to a base line position by using the eluent, collecting antibody peaks, dialyzing the antibody by using a PBS buffer solution, determining the antibody concentration by using a BCA protein quantitative kit, subpackaging and storing the antibody, and determining the titer by using indirect E L ISA.

TABLE 4T L R2(259-278aa) mouse subclone plate assay

1：Ab	131-0052
		5000	2.559
10000	1.921
		20000	1.601
40000	1.057
		80000	0.684
160000	0.518
		320000	0.347
BLANK	0.125

As shown in Table 4, the indirect E L ISA method was used to determine the titer of the purified T L R2(259-278aa) mouse monoclonal antibody, and the results showed that the antibody titers all reached 32 ten thousand and above.

Example 2

Experiment for preventing brucella from infecting macrophage by neutralizing antibody

1) Inoculating Brucella vaccine strain M5-90 into Brucella agar culture medium, sealing the opening, and placing in 10% CO₂Culturing in an incubator at 37 ℃, performing inverted culture for 3-5 days until a single colony grows in a solid culture medium, selecting the single colony to culture in a Brookfield broth liquid culture medium, culturing for 36 hours in a shaker at 37 ℃, collecting bacterial liquid, washing cells for 3 times by using PBS (phosphate buffer solution), centrifuging for 5min at 5000rpm by using a centrifuge, then re-suspending the bacterial liquid by using the PBS, and calculating the concentration of the bacteria by using a counter.

2) Macrophages were cultured using DMEM containing 10% FBS, and then the cells were seeded in 24-well plates (cell number per well 1 × 10)⁵) First, 100. mu.g of T L R2 antibody (259-278aa) was added (control group was replaced with ordinary IgG antibody) and the cells were incubated for 4 hours, then Brucella was added to infect the cells for 1 hour in a ratio of the number of bacteria to the number of cells of 50:1, the macrophages were thoroughly washed with PBS to remove extracellular Brucella, and then DMEM containing 10% FBS was added to continue culturing the macrophages for 24 hours.

3) Lysing cells on a culture plate with cell lysate, collecting cell lysate, diluting the cell lysate in a gradient of 1:10, 1:100, 1:1000, 1:10000 and 1:100000, mixing, uniformly coating 50 μ L volume of each dilution gradient in a Brinell agar medium, placing in 10% CO₂And culturing for 6h in an incubator at 37 ℃ and calculating the colony number of the Brucella.

The intracellular brucella survived in the experimental group and the control group are counted respectively at the time of no infection (0h) and at 4h, 24h, 48h and 72h after infection, and the results shown in Table 5 show that the infection efficiency of the brucella vaccine strain is remarkably reduced after the T L R2 (259-.

TABLE 5 CFU count of Brucella vaccine strain M5-90 infected human macrophage THP1

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A monoclonal antibody, which is an antibody targeting a gene sequence encoding amino acid 259-278 in the extracellular region of human T L R2.

2. The monoclonal antibody of claim 1, wherein the encoded 259-278 amino acid sequence is VKITDES L FQVMK LL NQISG.

3. A method of producing the monoclonal antibody of claim 1, comprising the steps of:

4. The method of claim 3, wherein the ratio of mouse spleen cells and mouse bone marrow cells in step 1 is 4: density of 1.

5. The method for producing a monoclonal antibody according to claim 3, wherein the obtained positive hybridoma cells are cloned twice in succession by limiting dilution, each clone is cultured in HT selective medium, and E L ISA screening is performed 7 days after cloning until the monoclonal cell positive rate is 100%.

6. Use of a monoclonal antibody according to any one of claims 1-2 in the preparation of a vaccine for inhibiting brucella.

7. The use of the monoclonal antibody of claim 6 in the preparation of a vaccine for inhibiting brucella, wherein the monoclonal antibody is used to inhibit infection of human macrophage THP-1 by a vaccine strain of brucella.