CN113234161B

CN113234161B - anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof

Info

Publication number: CN113234161B
Application number: CN202110703268.1A
Authority: CN
Inventors: 杨清海; 陈惠玲; 王小亚; 周洪辉
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-06-24
Filing date: 2021-06-24
Publication date: 2022-06-07
Anticipated expiration: 2041-06-24
Also published as: CN113234161A

Abstract

The invention relates to a monoclonal antibody capable of identifying human CD3 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. In the technical scheme, 1-65 amino acids at the C terminal of the CD3 protein are selected as antigen peptides to perform Codon optimization to obtain a gene fragment suitable for being expressed in Escherichia coli Codon plus, and the finally obtained recombinant protein comprises a TRX protein tag, a CD3 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 33H1 capable of efficiently secreting the anti-CD 3 protein monoclonal antibody and the anti-CD 3 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CD3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-CD 3 protein monoclonal antibody and cell strain, preparation method and application thereof

Technical Field

The invention relates to the field of biomedical engineering, in particular to an anti-CD 3 protein monoclonal antibody and a cell strain, a preparation method and application thereof.

Background

Immune cells, especially T cells, are in the core position in the whole process of immune response, and a plurality of CD molecules, such as CD3, CD4, CD8, CD28 and the like, exist on the surfaces of the T cells, and are widely involved in the whole process of immune effects, such as antigen recognition, activation and proliferation or incapacity, apoptosis, foreign antigen elimination or self-antigen tolerance, and the like of the T cells, among the CD molecules, CD3 molecules are particularly important, form a TCR-CD3 complex with T Cell Receptors (TCR) on the surfaces of the T cells, and have extremely important roles in the processes of T cell antigen recognition and immune response generation.

The CD3 molecule is distributed on the surface of mature T lymphocyte, is an important differentiation antigen on the T cell membrane, is one of the specific markers of T cell, and has 5.1 × 104 CD3 binding sites per T cell on average. The CD3 molecule is shown to be composed of at least five polypeptide chains, gamma, delta, epsilon, zeta, eta, which are tightly associated with the T cell antigen receptor (TCR) in a non-covalent manner. The main functions of the CD3 molecule are: stabilizing the TCR structure; transmitting a T cell activation signal.

CD3 plays an important role in the auxiliary diagnosis and differential diagnosis of T cell lymphoma. Lymphomas are a group of malignant tumors originated from lymph nodes or other lymphoid tissues and can be divided into Hodgkin Disease (HD) and non-Hodgkin lymphoma (NHL), the tumorous hyperplasia of lymphocytes and (or) histiocytes can be seen histologically, the clinical symptoms are most typical of painless lymphadenectasis, hepatosplenomegaly, and cachexia, fever and anemia at the late stage. T cell-derived lymphomas belong to the non-hodgkin lymphomas. Regarding the distribution of types, non-Hodgkin lymphoma is common in China, accounting for 95.1 percent and Hodgkin disease accounting for 4.9 percent. The incidence rate of Hodgkin's disease in China is low, which is close to 7.6% of that in Japan, but is common in European and American countries, and can reach as high as 45%. In the pathological types of non-Hodgkin lymphoma, the follicular form is rare in China, the incidence rate of T cell lymphoma and extranodal lymphoma is high, Japanese B cells account for 44.1 percent, more than 50 percent of the T cell source belongs to T cell sources, the incidence rate of the national and Japanese lymphomas is close, and European and American lymphomas are mostly derived from the B cells. non-Hodgkin lymphoma is more common than Hodgkin disease, can occur in all age groups, has the minimum attack age of 3 months and the maximum attack age of 82 years, and has the adult period of 2.7 in proportion to male and female: 1, 9-13 parts of children: 1.

clinically, the immunohistochemistry of CD3 is mainly used for the auxiliary diagnosis of T cell-derived lymphoma, and the differential diagnosis of T cell lymphoma, B cell lymphoma, malignant melanoma, and poorly differentiated cancer.

Disclosure of Invention

The invention provides a monoclonal antibody for resisting CD3 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.2, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3.

Further, the monoclonal antibody specifically recognizes the CD3 protein.

Further, the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 21980.

The inventor also provides a preparation method of the anti-CD 3 protein monoclonal antibody, wherein the antigen used for immunizing mice is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a TRX protein tag, a CD3 protein fragment and a His protein tag.

Furthermore, the CD3 protein fragment is the 23 rd to 126 th amino acid fragment of the CD3 protein, and the amino acid sequence of the CD3 protein fragment is the amino acid sequence shown in SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-CD 3 protein molecules, wherein the cell strain is a mouse hybridoma cell strain 33H1, the cell strain is preserved in China general microbiological culture Collection center (CGMCC NO 21980) at 3 months and 24 days in 2021, and the accession number is CGMCC NO 21980, and the microbial research institute of China academy of sciences No.3 of the North China West Lu No.1 Hopkins of the Korean district in Beijing.

The inventor also provides the application of the monoclonal antibody in immunodetection of the CD3 protein.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor finally provides a CD3 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence of which the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 4; the monoclonal antibody of anti-CD 3 protein with the amino acid sequence of the light chain variable region as shown in SEQ ID No.5 as the effective component.

Different from the prior art, the invention has the beneficial technical effects that: in the technical scheme, 23-126 amino acids at the C terminal of the CD3 protein are selected as antigen peptides to perform Codon optimization to obtain a gene fragment suitable for being expressed in Escherichia coli Codon plus, and the finally obtained recombinant protein comprises a TRX protein tag, a CD3 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain 33H1 capable of efficiently secreting the anti-CD 3 protein monoclonal antibody and the anti-CD 3 protein monoclonal antibody secreted by the cell strain are obtained through cell fusion, screening and subcloning. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing the CD3 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing the results of immunohistochemical staining of T cell lymphoma; the left is CD3 secreted by 33H1, and the right is commercial CD 3.

FIG. 2 is a comparison graph of immunohistochemical staining results of tonsils; the left is CD3 secreted by 33H1, and the right is commercial CD 3.

Detailed Description

To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.

EXAMPLE 1 preparation of recombinant CD3 protein fragment

First, gene optimization and synthesis

CD3 was directly optimized into a gene fragment suitable for expression in Codon plus of E.coli by selecting a protein fragment with amino acids 23-126 according to the protein sequence with accession number P07766 in the Uniprot database. EcoR I and XhoI cleavage sites were added at the 5 'and 3' ends of the gene, respectively, during PCR.

And separating and recovering the PCR product through agarose gel electrophoresis, carrying out EcoR I and XhoI enzyme digestion on the recovered fusion protein gene and a plasmid vector Pet32a for expression respectively, carrying out electrophoresis recovery again, and connecting with T4 DNA ligase. And transforming the connecting product into escherichia coli competent cell Codon plus, selecting the clone on the plate for inoculation, and performing PCR identification on the bacterial liquid. And selecting clones with positive PCR results for sequencing analysis, and using the clones with completely correct sequences.

The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. The CD3 molecule was analyzed according to published sequences, based on structure on cell membrane, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and secondary structure, selecting a region suitable for soluble expression and good immunogenicity for recombinant expression, and selecting amino acid residues 23-126 of CD3 for codon optimization, with a molecular weight of about 31 kDa. The CD3 protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of a CD3 protein fragment with antigenicity and a protein tag for purifying the recombinant protein, wherein the protein tag is TRX and His.

II, protein expression and purification

The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1:100, kanamycin with the final concentration of 10 mug/mL is added, shaking culture is carried out at 37 ℃ until OD600 is 0.6-0.8, 1mmol/L IPTG is added, shaking culture is carried out at 16 ℃ for overnight, and the strain is collected and then is crushed by ultrasound. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 500mmol/L imidazole and SDS PAGE separation was carried out.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The recombinant protein of example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week-old female ICR mice (purchased from Beijing Wintolite laboratory animals technologies, Ltd.) were immunized and injected subcutaneously at 6 o' clock into the abdomen of each mouse at a dose of 60. mu.g/mouse. The booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma Co., F5506) at a dose of 30. mu.g/mouse. 7 days after 3 times of booster immunization, the titer of multiple antibodies of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450nm), the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by normal saline, and the dosage is 50 mu g/mouse.

Second, cell fusion

A mouse spleen cell suspension with qualified immunity is prepared aseptically, mixed with mouse myeloma cell sp2/0(ATCC number CRL-8287) at a ratio of 5:1, and centrifuged at 1500rpm for 5 min. The supernatant was discarded and the tube was placed in a 37 ℃ water bath, 1ml of PEG1500 (Roche) was added slowly over 1 minute, and the cells were agitated. After standing in warm water for 1min, 10ml of serum-free IMDM (Sigma Co.) was added, mixed well, and centrifuged at 1000rpm for 5 min. After discarding the supernatant, 10ml of serum (PAA) was added to the supernatant, the cells were carefully blown up, 5ml of thymocytes mixed with 10XHAT (Sigma) was added, and the mixture was mixed well. Then, 25ml of a semi-solid medium containing 2.1% nitrocellulose (Sigma) was added thereto, mixed well, and poured into 20 cell culture dishes. Placing the cell culture dish into a wet box, and adding 5% CO at 37 deg.C₂Culturing in an incubator.

Cloning and ELISA screening of positive hybridoma cells

The size and density of the clone cell mass are moderate 7 days after fusion, and the round, solid and large clone mass is sucked under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and the culture is carried out in a 5% CO2 incubator at 37 ℃. After 3 days, the cell mass was approximately 2/3 basal areas, and 100. mu.L of the supernatant was screened by ELISA using the immunogen and the synthetic polypeptide, respectively. Positive clones were completely replaced and 200. mu.L of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, a second ELISA screening was performed and positive clones were transferred to 24-well plates previously prepared in medium (containing feeder cells and HT). After five days, 100 μ l of supernatant was subjected to a third ELISA screening, and the positive clones were transferred to 6-well plates and cell culture flasks successively for expanded culture and frozen.

EXAMPLE 3 preparation of monoclonal antibody by ascites Induction method

First, preparation of ascites

Cells in logarithmic growth phase were washed with serum-free medium and suspended, and counted at about 5X 10⁵And 1 ml. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites collection was started 7 days later. The ascites fluid taken out was centrifuged at 4000rpm at 4 ℃ for 10 min. The ascites fluid in the middle is carefully aspirated and collected in a centrifuge tube and stored at 4 ℃ or-20 ℃.

Secondly, purification of monoclonal antibody

Antibodies were purified from ascites fluid by HiTrap rProtein A FF (GE) affinity chromatography as described. Purity was assessed on SDS-PAGE gels and concentration was determined by Bradford method. Purified antibody was stored at-20 ℃.

EXAMPLE 4 monoclonal antibody characterization

First, subclass identification

Coated goat anti-mouse IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.) was diluted to 0.5. mu.g/ml with 100. mu.l/well at 4 ℃ overnight in 100mM PBS (pH 7.4). The liquid was decanted, washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. mu.l of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37 ℃ for 1 h. The liquid was decanted and washed 3 times with PBS-T. 0.1ml of hybridoma supernatant was added to each well and incubated at 37 ℃ for 1 hour. The decanted solution was washed 3 times with PBS-T. Using a confining liquid 1: 1000 dilution of HRP-labeled goat anti-mouse (κ, λ) antibody or 1: HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibodies (Southern Biotech) were diluted 2000 by 0.1ml per well and added to the appropriate wells, followed by incubation at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Add 50. mu.l of 0.15% ABTS (Southern Biotech) and 0.03% H per well₂O₂The reaction was performed in the citric acid buffer (pH4.0), and the OD value at a wavelength of 405nm was measured within 10 to 20 min.

The results show that the monoclonal antibody of the invention is a murine monoclonal antibody of the IgG2a kappa type.

Second, determination of affinity constant

The recombinant CD3 protein prepared in example 1 was coated at a concentration of 2. mu.g/ml, 100. mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. mu.l of blocking solution to each well and block for 2h at 37 ℃ and wash 3 times with PBS-T. The monoclonal antibody purified in example 3 was prepared from 1: 200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h, and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1: 20000 dilution, 100 μ l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of a buffer containing 0.1% TMB (Sigma) and 0.03% H was added to each well₂O₂The reaction mixture was developed in the citric acid-phosphoric acid buffer for 10min, and 50. mu.l of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of OD value corresponding to the dilution factor of the antibody, finding the dilution factor A corresponding to half of the maximum binding OD value, and calculating the affinity constant of the antibody to be 1.92 multiplied by 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The CD3 recombinant protein prepared in example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein bands were transferred to PVDF membranes (Millipore) in a Bio-Rad electrotransfer system according to the conventional method. The membrane was placed in TBS-T blocking solution containing 5% skimmed milk powder overnight at 4 ℃. Monoclonal antibodies to the antibody secreted by the 33H1 hybridoma (1: 1000 dilution) were added and incubated overnight at 4 ℃. After washing the membrane with TBS-T, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. Washing the membrane with TBST again, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad). Example 5 immunohistochemical tissue chip staining and identification

Chip preparation process

HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mold, putting the mold into an oven at 68 ℃ for 10 minutes to enable the tissue core and the wax of the receptor wax block to be fused into a whole, then slightly taking the mold out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, putting the mold into a refrigerator at-20 ℃ for freezing for 6 minutes, taking the tissue chip wax block out of the mold, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming, continuously slicing, setting the thickness to be 3 mu m, floating the continuous slices in 40% alcohol, naturally unfolding, transferring the separated slices into warm water at 50 ℃ for 30 seconds, pasting the slices with a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.

IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in a gradient of 85% (3 min) -95% (3 min) -100% (3 min) alcohol, finally xylene was transparent for 3 min, and the gel was blocked with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

Data statistics

1. Tumor tissue chip detection results:

the present antibody CD3(33H1) and the commercial antibody CD3(SP7) were simultaneously tested in 30 cases of T-cell lymphoma, and the test results were compared.

Immunohistochemical results for CD3 were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:

the result shows that the monoclonal antibody of the anti-CD 3 protein secreted by the 33H1 cell strain has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate is equivalent to that of a commercial antibody, but the partial positive intensity is higher than that of the commercial antibody, which indicates that the CD3 secreted by the 33H1 cell strain is more sensitive and effectively avoids false negative results.

FIG. 1 is a graph comparing the results of immunohistochemical staining of T cell lymphomas (33H 1-secreted CD3 on the left and commercial CD3 on the right).

2. Detection results of the normal tissue chip:

the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

The antibody (33H1) and the commercial antibody are synchronously detected on a normal tissue chip, and the negative and positive detection results are consistent, which shows that the specificity of the antibody on the normal tissue is equivalent to that of the commercial antibody.

FIG. 2 is a comparison of the results of immunohistochemical staining of tonsils (33H 1-secreted CD3 on the left and commercial CD3 on the right).

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising … …" or "comprising … …" does not exclude the presence of additional elements in a process, method, article, or terminal that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.

It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.

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Claims

1. The monoclonal antibody for resisting the CD3 protein is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID NO. 4; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 5.

2. The monoclonal antibody according to claim 1, wherein the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.2, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 3.

3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes the CD3 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG2a kappa subtype monoclonal antibody.

5. The monoclonal antibody of claim 1, which is produced by a hybridoma cell line with a collection number of CGMCC NO 21980.

6. A hybridoma cell strain capable of secreting anti-CD 3 protein molecules is a mouse hybridoma cell strain 33H1, is preserved in China general microbiological culture Collection center (CGMCC), and has the preservation number: CGMCC NO 21980.

7. Use of the monoclonal antibody of any one of claims 1-5 in the preparation of a CD3 protein immunodetection reagent.

8. An immunohistochemical detection reagent for CD3 protein, comprising the anti-CD 3 monoclonal antibody according to claim 1 as an active ingredient.