CN107083369B

CN107083369B - Anti-tree shrew CD3 molecular monoclonal antibody, hybridoma cell strain secreting antibody and application

Info

Publication number: CN107083369B
Application number: CN201710242349.XA
Authority: CN
Inventors: 徐靖雯; 董少忠; 张雪梅; 吴忠香; 蒋曦; 朱文兵; 宋杰; 李慧; 巩蔚; 严丽蔚
Original assignee: Institute of Medical Biology of CAMS and PUMC
Current assignee: Institute of Medical Biology of CAMS and PUMC
Priority date: 2017-04-14
Filing date: 2017-04-14
Publication date: 2020-10-09
Anticipated expiration: 2037-04-14
Also published as: CN107083369A

Abstract

The invention relates to a monoclonal antibody against tree shrew CD3 molecules, a hybridoma cell strain secreting the monoclonal antibody and application, and belongs to the technical field of molecular biology. The hybridoma cell strain is preserved in a China center for type culture collection, is named as hybridoma cell strain TSCD3-77B2 with the preservation number of CCTCC NO: C201741, and is preserved in eight paths of Wuhan university No. 299 in Wuhan district, Wuhan city, Hubei province. The monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity and strong affinity with natural antigens, can be used for detecting CD3 molecules on the surfaces of lymphocytes of natural tree shrew, and has better sensitivity and specificity.

Description

Anti-tree shrew CD3 molecular monoclonal antibody, hybridoma cell strain secreting antibody and application

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a monoclonal antibody, in particular to a hybridoma cell strain for generating a specific anti-tree-shrew CD3 molecular antibody, an anti-tree-shrew CD3 molecular antibody, and application of the antibodies.

Background

Tree shrews (Tupaia belangeri) are small animals of climbing shrews of mammalians living in tropical and subtropical regions, have a shape similar to squirrel, and are close relatives of primates. The tree shrews have the advantages of small size, economy, easy obtaining, easy domestication, strong reproductive capacity, low feeding and management cost, susceptibility to human viruses and the like, are often used for replacing or reducing the use of some non-human primates, and are therefore very early used as animal models of diseases of primates (including human beings). In recent years, the research shows that the compound has wide application in the research of diseases such as hepatitis C, hepatitis B, hand-foot-and-mouth diseases, liver cancer, diabetes, tumor and the like.

CD3 molecule: CD3 is a differentiated antigen expressed on the surface of all T lymphocytes that primarily mediates transduction of T cell activation signals. Plays an important role in anti-infection immunity of the body immune system. The CD3 molecule and a T cell antigen receptor (Tcell receptor) form a stable TCR-CD3 complex, the extracellular region of the CD3 molecule recognizes and combines with a major histocompatibility complex II molecule, and the combination stability of the T cell antigen receptor (TCR) and an MHC molecule is enhanced; the intracellular domain enhances the activation signal transduced by leukocyte CD3, thereby participating in and regulating the activation of the immune system. The number index of CD3 positive lymphocyte population is an important index for measuring the cellular immunity of the organism.

However, the research on the molecular aspect of the tree shrew CD3 is only reported at present, no available antibody is available for detecting the molecular level of the tree shrew CD3, the evaluation of analyzing the cellular immunity condition of the tree shrew animal model through lymphocyte surface molecular detection is still blank, and the research and the application of the tree shrew animal model are seriously hindered.

Disclosure of Invention

The monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity and strong affinity with natural antigens, and can be used for detecting CD3 molecules on the surfaces of lymphocytes of natural tree shrews.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a hybridoma cell strain secreting monoclonal antibodies against tree shrew CD3 molecules is preserved in China center for type culture collection and is named as hybridoma cell strain TSCD3-77B2 with the preservation number of CCTCC NO: C201741, and the preservation address is Wuhan university No. 299 in the Wuhan district, Wuhan city, Hubei province.

The invention also provides an anti-tree shrew CD3 molecular monoclonal antibody secreted by the hybridoma cell.

The invention also provides application of the anti-tree shrew CD3 molecular monoclonal antibody in preparation of a natural tree shrew lymphocyte surface CD3 molecular detection reagent or kit.

Further, the invention provides a flow cytometry detection reagent for CD3 molecules on the surfaces of lymphocytes of natural tree shrew, wherein the reagent comprises a monoclonal antibody which is labeled by Fluorescein Isothiocyanate (FITC) and is used for resisting the CD3 molecules of the tree shrew.

Further, the invention provides a western-blot detection reagent for CD3 molecules on the surfaces of natural tree shrew lymphocytes, wherein the reagent comprises a Horse Radish Peroxidase (HRP) labeled monoclonal antibody for resisting the tree shrew CD3 molecules.

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

the monoclonal antibody secreted by the hybridoma cell strain has the advantages of high titer, good specificity and strong affinity with natural antigens, and can be used for detecting CD3 molecules on the surfaces of lymphocytes of natural tree shrew.

The detection antibody established based on the method can effectively identify the CD3 molecule of the natural conformation on the surface of the tree shrew lymphocyte in a flow cell analysis experiment, and has better sensitivity and specificity. Number 10⁶Individual peripheral blood lymphocytes, detectable using FITC-labeled CD3 monoclonal antibody described aboveTree shrew lymphocyte surface CD3⁺The percentage of cells of (a) is about 89.5%.

The established western-blot detection reagent for the tree shrew CD3 protein can be used for analyzing the expression condition of CD3 molecules on the surface of the tree shrew peripheral blood lymphocytes, and the HRP-labeled CD3 monoclonal antibody is applied to detect the visible and clear CD3 molecular immunoblotting strip of 0.5ug of tree shrew lymphocyte lysis protein samples at the lowest.

Drawings

FIG. 1 is an electrophoresis diagram of expression and purification SDS-PAG E of GST tag and tree shrew CD3 fusion protein GST-TSCD3, wherein 1 is a protein molecular weight Marker, and 2 is a prokaryotic expression recombinant protein GST-TSCD3 after affinity purification;

FIG. 2 is an expression and purification SDS-PAGE electrophoresis picture of HIS label and tree shrew CD3 fusion protein HIS-TSCD3, wherein 1 is a protein molecular weight Marker, and 2 is a supernatant of an escherichia coli liquid expressing recombinant protein HIS-TSCD3 after ultrasonic treatment; 3 is recombinant protein HIS-TSCD3 after affinity purification;

FIG. 3 is a SDS-PAGE electrophoresis of the purified monoclonal antibody MAb-TSCD3-77B2, wherein 1 is a protein molecular weight Marker, and 2 is a MAb-TSCD3-77B2 sample after ProteinA purification elution and ultrafiltration concentration;

FIG. 4 is a western-blot diagram of monoclonal antibody MAb-TSCD3-77B2 recognizing CD3 protein in tree shrew lymphocytes, wherein 1 is 0.25 μ g of tree shrew PBMC cleavage protein, and 2 is 0.5 μ g of tree shrew PBMC cleavage protein; 3 is 1 mug of tree shrew PBMC cleavage protein; 4 is 2 ug of tree shrew PBMC cleavage protein, 5 is 3 ug of tree shrew PBMC cleavage protein.

FIG. 5 is a diagram of flow cytometry analysis of FITC-labeled monoclonal antibody MAb-TSCD3-77B2 recognizing tree shrew lymphocyte surface CD3 molecules, wherein a is a Peripheral Blood Mononuclear Cell (PBMC) negative control; b is CD3 in PBMC⁺The lymphocyte of (4).

The hybridoma cell strain TSCD3-77B2 is built in China center for type culture Collection in 3.16.2017, has the preservation number of CCTCC NO: C201741, and has the preservation address of Wuhan university No. 299 in eight Wuhan district, Wuhan city, Hubei province.

Detailed Description

The present invention will be described in further detail with reference to examples.

It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available by purchase.

Firstly, establishing a hybridoma cell strain:

(1.1) construction of prokaryotic expression vectors pGEX-5X-TSCD3 and Pet3.0-TSCD3

In order to express tree shrew CD3 molecular protein in an escherichia coli expression system, peripheral blood is collected from tree shrew tail veins, lymphocytes obtained by separation are separated by lymphocyte separation fluid (purchased from AXIS-SHIELD), RNA is extracted by TRI ZOL (heaven), coding gene fragments of tree shrew CD3 molecules are obtained by reverse transcription PCR (purchased from Baobao), T4 ligase (purchased from Baobao) is connected to an expression vector pGEX-5X-1 to obtain a GST tag recombinant protein expression plasmid pGEX-5X-TSCD3, and the tree CD3 molecular gene is connected to an expression vector pet3.0 to obtain a HIS tag recombinant protein expression plasmid Pet3.0-TSCD 3. Wherein the DNA sequence of the tree shrew CD3 molecule is shown as SEQ ID NO. 1.

(1.2) expression of recombinant proteins GST-TSCD3 and HIS-TSCD3

The plasmids pGEX-5X-TSCD3 and Pet3.0-TSCD3 are transformed into prokaryotic expression host bacteria BL21, induced and expressed for 3 hours at 37 ℃, 8000 turns, centrifugates for 10 minutes to collect bacteria, suspends by affinity purified binding buffer solution, ultrasonically breaks the bacteria, centrifugates again, the centrifugation condition is the same as the above, takes the supernatant for affinity purification, and obtains recombinant protein GST-TSCD3 (figure 1) for immune animals and recombinant protein HIS-TSC D3 (figure 2) for screening and detecting.

(1.3) animal immunization: selecting Balb/C healthy mice (provided by animal center of institute of medical biology of Chinese academy of medical sciences) with the age of about 8 weeks, completely emulsifying prokaryotic expression fusion protein GST-TSCD3 with GST tag and equivalent Freund's complete adjuvant (immunization 1 st) or Freund's incomplete adjuvant (immunization 2 nd and 3 rd) (all adjuvants are purchased from sigma), and performing subcutaneous multipoint immunization on the back, wherein the amount of immune protein of each mouse is 100 mu g. The immunization procedure was carried out on day 1, day 28 and day 56, respectively, and on day 59, spleen cells of mice were ground to white tissue and filtered through a 70-mesh cell screen to obtain spleen cells of mice.

(1.4) preparation of feeder cells: selecting a 12-week-old Balb/C healthy mouse (provided by animal center of institute of medical and biology, national academy of medical sciences), breaking neck, sterilizing with 75% ethanol for 5 min, cutting off abdominal skin with aseptic scissors, slowly peeling off, exposing peritoneum, injecting 10ml of precooled sucrose solution with mass concentration of 16% into abdominal cavity of the mouse with aseptic injector, massaging abdominal part of the mouse for 5 min, extracting liquid in the abdominal cavity with injector, centrifuging for 1000 min, suspending supernatant with culture solution, counting, adjusting volume of the culture solution to make cell concentration be 10⁵About one/ml, then adding 100. mu.l per well into 96-well plate, standing at 37 deg.C and 5% CO₂Culturing under the condition for 1-2 days.

(1.5) fusion of mouse spleen cells and SP2/0 cells SP2/0 cells (purchased from ATCC) in logarithmic growth phase were mixed with the number of mouse spleen cells obtained in step (1.3) at a ratio of 1:10 in terms of cell number, and hybridoma cells were obtained by the polyethylene glycol (PEG) (purchased from sigma) method after which the hybridoma cells were added to the feeder cell-containing 96-well plate obtained in step (1.4), and cultured medium 1640 containing 1 × HAT (purchased from sigma) and 20% (V/V) fetal bovine serum 1640 at 37 ℃ in 5% CO₂Culturing under the condition.

(1.6) screening of hybridoma cell lines: the hybridoma obtained in step (1.5) was cultured in 1640 medium containing 1 XHAT, 20% (V/V) fetal bovine serum (purchased from Biological Industries, BI) (purchased from Corning) for 10 days, and after culturing in 1640 medium containing 1 XHT (purchased from sigma), 20% (V/V) fetal bovine serum for 5 days, the hybridoma was cultured in 1640 medium containing 20% (V/V) fetal bovine serum for expansion. And (3) taking hybridoma cell supernatant, detecting antibody titer by using an ELI SA plate coated with prokaryotic expression recombinant protein HIS-TSCD3 protein, and taking unfused SP2/0 cell culture supernatant as a negative control. And selecting cell holes with the detection OD value higher than 6 times of negative value for further subcloning.

(1.7) selecting the cell wells with good growth state and detection OD value higher than 6 times negative on the 96-well plate in the step (1.6), sucking the cells in each well to a sample adding groove, diluting the cells to 20ml by using 1640 culture medium containing 20% (V/V) fetal calf serum, uniformly adding the diluted cells to a 96-well plate, wherein 200 mu l of the cells in each well are added at 37 ℃ and 5% CO in the 1640 culture medium₂And (3) culturing for 5 days under the condition, detecting again, selecting a cell hole with the OD value being 6 times higher than the negative value and the cell growth state being good, cloning to another 96-well plate, and repeating the steps for three to four times until the OD of the whole 96-well plate is 6 times higher than the negative hole. And selecting cells in one hole with good cell growth state, gradually carrying out amplification culture, and freezing and storing the cells in liquid nitrogen.

Secondly, preparing monoclonal antibody ascites in large scale:

injecting the positive (OD value is 6 times higher than that of the negative hole) clone cells obtained by the enlarged culture in the step (1.7) into 10-week-old Balb/C healthy mice sensitized by paraffin oil in advance, wherein the cell injection amount of each mouse is 10⁶-10⁷10-14 days later, ascites is collected, and the titer is detected>1:3200 ascites sample is split-packaged and frozen.

Thirdly, purifying the antibody:

and (3) purifying the mouse ascites obtained in the second step by a proteinA column (purchased from holotype gold) to obtain a monoclonal antibody MAb-TSCD3-77B2 of the mouse anti-tree shrew CD3, concentrating the monoclonal antibody obtained in the step (figure 3) to 1mg/ml by an ultrafiltration centrifugal tube, and subpackaging and freezing for later use.

Fourthly, labeling of the monoclonal antibody:

and (3) Labeling the monoclonal Antibody MAb-TS CD3-77B2 of the purified and concentrated mouse anti-tree shrew CD3 obtained in the third step with a fluorescent dye FITC according to the instruction operation of a Labeling Kit EZLabel Antibody FITC Labeling Kit (Biovision), adding glycerol with the same volume after the reaction is finished, mixing uniformly, and subpackaging at-20 ℃ for storage.

And (3) labeling the monoclonal antibody MAb-TS CD3-77B2 of the purified and concentrated mouse anti-tree shrew CD3, which is obtained in the third step, with HRP (horse radish peroxidase) according to the operation of an HRP labeled antibody kit (Proteitech) instruction, adding glycerol with the same volume after the reaction is finished, mixing uniformly, and subpackaging at-20 ℃ for storage.

The product of the invention comprises the following components and characteristics:

after a mouse is immunized by tree shrew CD3 protein (GST-TSCD3) with a GST tag expressed by a prokaryotic expression system, a cell strain TSCD3-77B2 which secretes a specific CD3 monoclonal antibody MAb-TSCD3-77B2 positive can be obtained, and natural tree shrew lymphocyte surface CD3 molecules can be specifically identified.

The antibody application comprises monoclonal antibody MAb-TSCD3-77B2 marked by fluorescent dye FITC, and is mainly applied to flow cytometry analysis.

The antibody application also comprises a horseradish peroxidase (HRP) -labeled monoclonal antibody MAb-TSCD3-77B2, and the detection of the western-blot is mainly applied.

The application method comprises the following steps:

firstly, a flow cytometry detection method comprises the following steps:

1. each 100. mu.L of single cell suspension isolated from peripheral blood, about 1 × 10⁶(ii) individual cells;

2. sample preparation: adding 4 mul of monoclonal antibody which is well labeled by a fluorescent dye FITC and is used for resisting the tree shrew CD3 molecules and has the concentration of 0.5 mug/mul into one part of cells;

blank: taking a cell without adding the antibody;

3. reacting for 1-2 hours at room temperature in a dark place;

4. adding 1mL of PBS into each part, washing the cells gently once, centrifuging at 2000rpm/min for 10min, and removing the supernatant;

5. adding 300 mu L PBS each to resuspend the cells, and detecting and analyzing by a flow cytometer.

6. The results are shown in FIG. 5, with a number of 10⁶Peripheral blood lymphocytes, the tree shrew lymphocyte surface CD3 could be detected by using the above C D3 monoclonal antibody labeled by FITC⁺The percentage of cells of (a) is about 89.5%.

(II) the use method of western blot Werstern-blot detection of the tree shrew CD3 is as follows:

1. sample processing and loading and electrophoresis

5 xSDS-PAGE protein loading buffer [250Mm Tris-HCl (pH6.8), 10% (W/V) SDS, 0.5% (W/V) BPB, 50% (V/V) glycerol, 5% (W/V) beta-mercaptoethanol ] is added to the collected lymphocyte protein sample or recombinant protein sample in proportion, and the mixture is heated at 95 ℃ for 5 minutes to fully denature the protein. And cooling to room temperature, and directly loading the protein sample into the SDS-PAGE gel sample loading hole. 80V 25 min, 150V 60 min electrophoresis.

2. Transfer film (Transfer)

The electrophoresed proteins were transferred to PVDF membrane (purchased from Millipore) according to the instructions of the membrane transfer apparatus using a semidry membrane transfer apparatus (BIO-RAD) with a membrane transfer current of 100mA and a membrane transfer time of 45 minutes.

3. Seal (Blocking)

Immediately after the completion of the membrane transfer, the PVDF membrane subjected to the membrane transfer was placed in 5% (W/V) skimmed milk powder TBS [20mM Tris-HCl, 500mM NaCl (pH7.5) ] prepared in advance, and the mixture was gently shaken on a shaker and sealed at room temperature for 60 minutes.

4. Primary antibody incubation (Primary antibody incubation)

HRP-labeled MAb-TSCD3-77B2 (500. mu.g/ml) was diluted with TBS at a ratio of 1:1000, and the blocked PVDF membrane was incubated on a side shaker at room temperature for one hour or overnight at 4 ℃.

5. Washing membrane

Discarding the primary antibody diluent, placing the primary antibody incubated membrane into a membrane washing box (purchased from Millipore), adding 10ml of membrane washing solution TBST [20mM Tris-HCl, 500mM NaCl (pH7.5), 0.01% Tween-20(V/V) ]10ml, shaking on a side shaking table, changing new TBST after 10 minutes, and repeating for 4 times;

6. protein Detection (Detection of proteins)

ECL (from Millipore) reagent was added as per the instructions, and the film was pressed in the dark room and developed with developer fixing solution. As a result, as shown in FIG. 4, a clear CD3 molecular immunoblot band was observed in the tree shrew lymphocyte lysate sample at a minimum of 0.5 ug.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

SEQUENCE LISTING

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Claims

1. A hybridoma cell strain secreting monoclonal antibodies against tree shrew CD3 molecules is characterized by being preserved in China center for type culture collection for establishment of strains, wherein the preservation name is hybridoma cell strain TSCD3-77B2, the preservation number is CCTCC NO: C201741, and the preservation address is Wuhan university No. eight in Wuchang region of Wuhan city, Hubei province.

2. An anti-tree shrew CD3 molecular monoclonal antibody secreted by the hybridoma cells of claim 1.

3. The use of the monoclonal antibody against tree shrew CD3 molecules of claim 2 in the preparation of a detection reagent or a kit for the natural tree shrew lymphocyte surface CD3 molecules.

4. A flow cytometry detection reagent for CD3 molecules on the surfaces of lymphocytes of natural tree shrew, which is characterized in that the reagent comprises the anti-tree shrew CD3 molecular monoclonal antibody which is marked by fluorescein isothiocyanate and is used for resisting the tree shrew.

5. A western-blot detection reagent for CD3 molecules on the surfaces of lymphocytes of natural tree shrew, which is characterized in that the reagent comprises a monoclonal antibody against the tree shrew CD3 molecules, wherein the monoclonal antibody is marked by horseradish peroxidase.