CN113845592A

CN113845592A - anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application

Info

Publication number: CN113845592A
Application number: CN202111134257.2A
Authority: CN
Inventors: 赵普; 杨清海; 陈惠玲; 王小亚; 周洪辉; 程本亮; 陈昌星; 林婷; 李萍
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2021-12-28
Anticipated expiration: 2041-09-27
Also published as: CN113845592B

Abstract

The invention relates to a monoclonal antibody capable of identifying human CK5/6 antigen, a secretory cell strain, a preparation method thereof and application thereof in immunoassay. The technical scheme selects the 488-590 site amino acid at the C terminal of the CK5/6 protein as an antigen peptide to be optimized to form a gene fragment suitable for being expressed in escherichia coli BL21, and the finally obtained recombinant protein comprises a TRX protein tag, a CK5/6 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain AbM58019-7H4 capable of efficiently secreting the anti-CK 5/6 protein monoclonal antibody and the anti-CK 5/6 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 CK5/6 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Description

anti-CK 5/6 protein monoclonal antibody, cell strain thereof, preparation method and application

Technical Field

The invention relates to the field of biomedical engineering, in particular to a monoclonal antibody for resisting CK5/6 protein, a cell strain, a preparation method and application thereof.

Background

The Cytokeratin (CKs) family is a group of cytoskeletal intermediate filaments distributed within epithelial cells, the expression of which is tissue and organ specific, and is mainly expressed in epithelial cells, with a total of 20 members. Cytokeratin5/6 (cytokeratin5/6, CK5/6) is a high molecular weight cytokeratin, a high molecular weight acidic polypeptide substance, comprising two molecules, CK5 and CK6, mainly expressed in squamous epithelium of epithelium and mucosa, such as squamous epithelium of lung, basal muscle epithelial layer of mammary gland, prostate gland and salivary gland. Its role, like other cytokeratins, is to maintain the morphological integrity of epithelial cells. The expression of CKs in tumor tissue can be detected to determine whether the tumor is of epithelial origin or whether there is a deficiency in epithelial components. Most of the single-layer glandular epithelium does not express CK5/6, and has good differential diagnosis effect on squamous carcinoma and adenocarcinoma.

CK5/6 protein is often expressed on the basal cell layer of normal mammary duct. CK5/6 has also been shown to be a characteristic marker of basal cell-like breast cancer. The literature reports that the survival time of breast cancer patients expressing CK5/6 is relatively short. There are also studies reporting that CK5/6 has been considered as an independent prognostic indicator for breast cancer. It is also reported in the literature that the expression of CK5/6 positive cells in the peripheral blood of breast cancer patients is related to the stage of breast cancer, and the CK5/6 positive cells can be used as a reference index for clinical monitoring and prognosis judgment. CK5/6 is currently used as a characteristic marker of basal cell-like breast cancer in molecular typing.

Disclosure of Invention

The inventor provides an anti-CK 5/6 protein monoclonal antibody, 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 CK5/6 protein.

Further, the monoclonal antibody is a mouse IgG2b kappa subtype monoclonal antibody.

Furthermore, the monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 21982.

The inventor also provides a preparation method of the anti-CK 5/6 protein monoclonal antibody, an antigen used for immunizing a mouse is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a TRX protein tag, a CK5/6 protein fragment and a His protein tag.

Further, the CK5/6 protein fragment is an amino acid fragment at position 488-590 of the CK5/6 protein, and the amino acid sequence of the fragment is the amino acid sequence shown as SEQ ID NO. 1.

The inventor also provides a hybridoma cell strain secreting anti-CK 5/6 protein molecules, wherein the cell strain is a mouse hybridoma cell strain AbM58019-7H4, the cell strain is preserved in China general microbiological culture Collection center (CGMCC) at 24 months in 2021, the preservation number is CGMCC NO 21982, and the address is the institute of microbiology of China academy of sciences No.3 of the West Lu No.1 of the sunward area, Beijing.

The inventor also provides the application of the monoclonal antibody in CK5/6 protein immunoassay.

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

The inventor finally provides a CK5/6 protein immunohistochemical detection reagent, wherein the immunohistochemical detection reagent contains an amino acid sequence shown as SEQ ID No.4 in the amino acid sequence of a heavy chain variable region; the monoclonal antibody of anti-CK 5/6 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, the 488-590 site amino acid at the C-terminal end of the CK5/6 protein is selected as an antigen peptide and optimized into a gene fragment suitable for being expressed in escherichia coli BL21, and the finally obtained recombinant protein comprises a TRX protein tag, a CK5/6 protein fragment and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and a mouse hybridoma cell strain AbM58019-7H4 capable of efficiently secreting the anti-CK 5/6 protein monoclonal antibody and the anti-CK 5/6 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 CK5/6 protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph comparing immunohistochemical staining results for esophageal squamous cell carcinoma; wherein the left part is CK5/6 secreted by AbM58019-7H4, and the right part is commercial CK 5/6.

FIG. 2 is a graph comparing the results of immunohistochemical staining of esophagus; wherein the left part is CK5/6 secreted by AbM58019-7H4, and the right part is commercial CK 5/6.

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 CK5/6 protein fragment

First, gene optimization and synthesis

According to the protein sequence with the accession number P13647 in the Uniprot database, the CK5/6 selects the protein fragment with the amino acid at position 488-590, and directly optimizes the protein fragment into the gene fragment suitable for being expressed in the Escherichia coli BL 21. 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 cells BL21, selecting clones on a plate for inoculation, and performing PCR identification on 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 CK5/6 molecule was analyzed according to published sequences, based on the structure, antigenicity, hydrophilicity and hydrophobicity of the constituent amino acids and secondary structure in the cytoplasm, a region suitable for soluble expression and good immunogenicity was selected for recombinant expression, and the amino acid residue at position 488-590 of CK5/6 was selected for codon optimization and recombinant expression, with a molecular weight of about 31 kDa. The CK5/6 protein is obtained by using prokaryotic expression gene sequence through sequence optimization design. The recombinant immunogen consists of an antigenic CK5/6 protein fragment and a protein tag for purifying 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 characterization of monoclonal antibodies

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 per well was added at 37 deg.CIncubate for 1 h. 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 at 2000 in 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 IgG2b kappa type.

Second, determination of affinity constant

CK5/6 recombinant protein prepared in example 1 was used at a coating 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 a 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 3.84 × 10 by using the following formula⁹。

Reaction specificity and application effect of monoclonal antibody

The CK5/6 recombinant protein prepared in example 1 was used for detecting 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 (1: 1000 dilution) to the antibody secreted by the AbM58019-7H4 hybridoma 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, adding diluted primary antibody (the dilution ratio of the antibody is designed according to the concentration of the antibody in the first dilution) dropwise, incubating at room temperature (25 ℃) for 1 hour, washing with PBS for 3X 3 minutes, adding secondary antibody dropwise, incubating at room temperature for 15-30 minutes, washing with PBSAnd 3X 3 minutes, throwing off 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 antibody CK5/6(AbM58019-7H4) and the commercially available antibody CK5/6(D5/16B4) were tested simultaneously on 35 multi-tumor tissue chips and the results were compared.

The immunohistochemical results of CK5/6 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-CK 5/6 protein secreted by the AbM58019-7H4 cell strain has accurate staining location, 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 CK5/6 secreted by the AbM58019-7H4 cell strain is higher in sensitivity, and the false negative result is effectively avoided.

FIG. 1 is a graph showing the comparison of immunohistochemical staining results for esophageal squamous carcinoma (CK 5/6 secreted by AbM58019-7H4 on the left, and CK5/6 commercially available 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 (AbM58019-7H4) and the commercial antibody were detected on a normal tissue chip synchronously, and the negative and positive detection results were consistent, indicating that the specificity of the antibody in normal tissues was equivalent to that of the commercial antibody.

FIG. 2 is a graph comparing the results of immunohistochemical staining of esophagus (CK 5/6 secreted by AbM58019-7H4 on the left, and CK5/6 commercially available 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 CK5/6 protein is characterized in that the amino acid sequence of a 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 CK5/6 protein.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a mouse IgG2b kappa subtype monoclonal antibody.

5. An anti-CK 5/6 monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 21982.

6. The preparation method of the anti-CK 5/6 protein monoclonal antibody is characterized in that an antigen used for immunizing a mouse is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode and comprises a TRX protein tag, a CK5/6 protein fragment and a His protein tag.

7. The method as claimed in claim 6, wherein the CK5/6 protein fragment is the amino acid fragment at position 488-590 of the CK5/6 protein, and the amino acid sequence is shown as SEQ ID NO. 1.

8. A hybridoma cell strain secreting anti-CK 5/6 protein molecules is a mouse hybridoma cell strain AbM58019-7H4, is deposited in the China general microbiological culture Collection center (CGMCC), and has the following preservation numbers: CGMCC NO 21982.

9. Use of the monoclonal antibody of any one of claims 1-5 in an immunoassay for the CK5/6 protein.

10. An immunohistochemical detection reagent for CK5/6 protein, comprising the anti-CK 5/6 protein monoclonal antibody of claim 1 as an active ingredient.