CN108467432B - Monoclonal antibody of anti-E-cadherin protein, cell strain, preparation method and application thereof - Google Patents

Abstract

The invention relates to a monoclonal antibody capable of detecting E-cadherin, a preparation method of a hybridoma cell strain 36C4 thereof and application of the monoclonal antibody. The antigen for preparing the antibody is a section of recombinant protein selected from E-cadherin protein, a mouse is immunized after the recombinant protein is expressed and purified by escherichia coli, hybridoma cell strains are screened by the recombinant protein and tumor cells, and finally the obtained antibody belongs to an IgG2b subtype. After extracting RNA from the 36C4 cell line obtained by screening, the sequence encoding the antibody variable region was amplified by reverse transcription using the cDNA obtained as a template. The antibodies of the invention can be used in immunoassays.

Description

Monoclonal antibody of anti-E-cadherin protein, cell strain, preparation method and application thereof

Technical Field

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

Background

E-Cadherin is a calcium ion-dependent cell adhesion protein (Ca)²⁺dependent Cell attachment family), also known as morula adhesion protein (Uvomorulin), L-CAM or Cell-CAM 120/80. There are two more protein members of this family, N-Cadherin and P-Cadherin. Different adhesion protein molecules have unique tissue distribution patterns in vivo, and the expression of the adhesion protein molecules varies with the growth and development state of cells. E-cadherin is a transmembrane epithelial protein that triggers intercellular adhesion, and its deletion is associated with tumor spread, where dysfunction in tumor cells may cause cell shedding and metastasis. The protein is generally membrane-stained and cytoplasmic-stained. The protein has reduced expression level in the diffuse bladder cancer, different tumors in the breast part, no expression in lobular carcinoma and Lobular Carcinoma In Situ (LCIS) of mammary gland, reduced expression in ductal adenocarcinoma, and positive in small cell carcinoma and Ductal Carcinoma In Situ (DCIS). There is no expression in signet-ring cell carcinomas (net-ring cell carcinoma) of the stomach, and in colon adenocarcinomas, a weakening of the membrane staining occurs compared to epithelial cells dislocated in adenomas.

In histopathology, breast infiltrating cancer is marked by the absence of a muscle epithelial cell layer surrounding the cancer foci. Immunohistochemistry is an important aid for the pathological diagnosis of early invasive breast cancer, and common markers of the myoepithelial cell layer are smooth myosin heavy chain (SMM-HC), actin binding protein (Calponin) and p 63. However, a single myoepithelial marker does not determine whether a small number of tiny cell masses in the stroma that lack a myoepithelial cell layer are cancer cell masses. The Miyayo adopts a method of double-staining an E-Cadherin antibody and a Calponin antibody to improve the diagnosis level of early invasive carcinoma of mammary gland (Miyayo. the application of immunohistochemical double-staining method in the pathological diagnosis of early invasive carcinoma of mammary gland. Zhengzhou university Master paper, 2016.)

The invention discloses an antibody chip capable of simultaneously detecting multiple cell adhesion factors (publication No. CN107328941A), which discloses an antibody chip capable of simultaneously detecting multiple cell adhesion factors, wherein the detection of E-cadherin is also included, and the E-cadherin protein detected by the method has no tissue and cell location information and is not suitable for the pathological diagnosis of most tumors. In addition, the antibody can also be used for enriching and purifying free tumor cells in the circulatory system, for example, the invention patent "an E-Cadherin, Cadherin-11, EpCAM multiple antibody immunomagnetic bead and its preparation method" (publication No. CN106432504A) uses a plurality of antibodies for enriching CTC cells in blood by a magnetic bead coupling method, and similarly, the antibody has no detection application of immunohistochemical pathology. In the invention patent "anti-E-Cadherin protein monoclonal antibody hybridoma cell and its produced anti-E-Cadherin monoclonal antibody and application" (application No. 201510703037.5) using immunohistochemistry as an application, a method for preparing a monoclonal antibody using HEK293 cell transfected with E-Cadherin gene as immunogen is described, the antibody can be used for immunohistochemistry and immunoblot detection, the immunogen is full-length gene, and in more than 30 members of Cadherin family, the similarity is very high, so the recognition specificity needs to be fully confirmed.

Various scientific commercial antibodies are available on the market. But the specificity and sensitivity and staining results for immunohistochemical detection were not sufficient. Murine mAb 4A2C7 available from Zymed, Thermo Fisher et al, 36B5 available from Leica Microsystems, and Sigma-Aldrich (MERCK), the rabbit mAb product HPA004812, are used for immunohistochemical analysis, but the results are different and still continue to improve sensitivity and specificity.

Disclosure of Invention

The invention provides an anti-E-cadherin monoclonal antibody which is produced by a hybridoma cell strain with the preservation number of CGMCC NO 15489. The cell strain is preserved in the China general microbiological culture Collection center in 2018, 3 and 9 months, and is classified and named as: the mouse hybridoma cell line has the preservation number: CGMCC NO 15489, applied to institute of microbiology of China academy of sciences No.3, Xilu No.1, Beijing, Chaoyang.

Further, the monoclonal antibody specifically recognizes the E-cadherin protein.

Further, the monoclonal antibody specifically recognizes the amino acid sequence shown as SEQ ID1 in the E-cadherin protein.

Further, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID3, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID 4.

Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown as SEQ ID 5; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown as SEQ ID 6.

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

The inventor also provides a preparation method of the anti-E-cadherin monoclonal antibody, amino acids 498 to 709 of the E-cadherin protein are selected as immunogens to carry out protein recombination, and the recombinant protein is recombined and expressed by escherichia coli. The immunogen is fused on both sides with 6 histidines as tags.

The inventor also provides a hybridoma cell strain secreting E-cadherin-resistant protein molecules, wherein the cell strain is a mouse hybridoma cell strain 36C4, and the cell strain is deposited in China general microbiological culture Collection center (CGMCC) 3, 9 and 2018, and is classified and named as: the mouse hybridoma cell line has the preservation number: CGMCC NO 15489, applied to institute of microbiology of China academy of sciences No.3, Xilu No.1, Beijing, Chaoyang.

The inventor also provides the application of the monoclonal antibody in the immunodetection of the E-cadherin protein.

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

Different from the prior art, the invention has the beneficial technical effects that:

(1) the monoclonal antibody obtained by the invention is secreted and generated by hybridoma 36C4, can specifically recognize recombinant expression and E-cadherin in human epithelial cells, and can specifically detect the expression condition of the E-cadherin in normal tissue epithelium, mammary epithelial cells and bladder cancer tissues.

(2) The hybridoma 36C4 obtained by the invention is an Ig2b subtype antibody, and has extremely strong specificity and sensitivity in combination with natural E-cadherin.

(3) The monoclonal antibody hybridoma 36C4 obtained by the invention takes an extracellular membrane-proximal region fragment, and artificially synthesizes a gene fragment as an immunogen after optimizing a codon sequence, the region has good surface accessibility, and appropriate secondary structure and amino acid composition characteristics, and the prepared antibody can meet the detection requirements of Immunohistochemistry (IHC), immunoblotting (Western blotting) and ELISA methods.

Drawings

FIG. 1 is a diagram showing alignment before and after optimization of the YE-cadherin gene fragment sequence; 1 is optimized sequence E-cadherin-F, 2 is partial sequence of original reference gene sequence NM-004360.4, the similarity of the two is 76.5%, and 150 bases in the sequence are optimized.

FIG. 2 is a graph showing the result of electrophoresis of a small expression of recombinant E-cadherin protein; m: molecular weight markers, 1-5: randomly picked 5 monoclonal strains;

FIG. 3: electrophoresis result chart of purified E-cadherin-F protein; m: molecular weight marker, E-cadherin-F: imidazole-eluted recombinant E-cadherin-F protein;

FIG. 4 is an immunoblot of purified mAb against E-cadherin protein; in the experiment, the concentration of the recombinant E-cadherin protein is 2 mug/ml, the total amount of the recombinant E-cadherin protein is 20ng, the concentration of the 67127-36C4 antibody is 1mg/ml, the recombinant E-cadherin protein is diluted at a ratio of 1:1000 for use, and the HRP enzyme is labeled to goat anti-mouse at a ratio of 1:5000 for use.

FIG. 5 is a graph comparing the staining results of breast cancer sample 1;

FIG. 6 is a graph showing a comparison of the staining results of breast cancer sample 2.

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 recombinant protein preparation:

the E-cadherin protein sequence with the number P12830 was selected from the Uniprot database (http:// www.uniprot.org) as a standard sequence, the sequence of which is shown in sequence listing ID1, compared to sequence differences with other proteins of the same family by BLAST (https:// BLAST. ncbi. nlm. nih. gov/BLAST. cgi) tools, and analyzed for secondary structure, antigenicity and surface accessibility with the Protean module of DNASTAR8.0 software (www.dnastar.com). Amino acids 498 to 709 of the E-cadherin protein were selected as recombinant proteins, and this region was adjacent to the extracellular region of the transmembrane region.

(1) Gene synthesis and cloning

In order to improve the expression yield, the DNA of the selected region was codon optimized, the nucleic acid fragment was obtained by means of gene synthesis (Biotechnology engineering (Shanghai) Co., Ltd.), BamHI and XhoI cleavage sites were added upstream and downstream of the coding sequence, respectively, for the convenience of cloning, the fragment was recovered by double cleavage with BamHI and XhoI, the fragment was ligated into the linearized expression vector pET30a (Novagen, now Merck) which had been similarly double cleaved, and the gene fragment cloned into the expression vector was named E-cadherin-F.

Factors which need to be comprehensively considered in the codon optimization process comprise the removal of enzyme cutting sites, the adjustment of GC content in a coding region, the replacement of rare codons of escherichia coli and the adjustment of low-preference codons into high-preference codons of a host, and the optimization of the codons can improve the yield and the stability of transcription products and can improve the expression level of proteins. The reference sequence of the E-cadherin protein in Genbank is NM-004360.4, and the sequence is optimized on the basis of the sequence. The optimized evaluation index is Codon Adaptability Index (CAI), the closer the index is to 1, the better the index is, the GC content of the sequence should be controlled between 30% and 70%, and the concatenation degree (CFD) of rare codons should be lower than 30%, the lower the index is, the better the index is. After sequence optimization, the codon adaptation index of the target sequence is improved from 0.61 to 0.87, the GC content is adjusted from 48.74% to 50.44%, and the CFD value is reduced from 10% to 0. The alignment results before and after sequence optimization are shown in figure 1, and the alignment graphs before and after YE-cadherin gene fragment sequence optimization. The expression yield of the synthetic gene is higher than that of the general gene, and the sequence optimization is shown to obtain beneficial effects.

The specific scheme is as follows:

double digestion of gene fragment

Digesting for 3h at 37 ℃, detecting the enzyme digestion product by electrophoresis, and recovering the enzyme digestion DNA fragment for connection.

Digesting for 3h at 37 ℃, detecting the enzyme digestion product by electrophoresis, and recovering the linearized pET30a vector fragment for connection.

Connection and transformation:

3. mu.l of linearized pET30a1 vector

Mu.l of the digested fragment

Ligase mixture (TaKaRa) 5. mu.l

Mixing, connecting at 16 ℃ overnight, taking out escherichia coli BL21 competent cells (Novagen, Merck), slowly thawing at-80 ℃, adding the connection product, thermally shocking at 42 ℃ for 90s after ice bath for 30min, adding 800 ul of nonresistant LB culture medium, culturing at 37 ℃ for 45min, centrifuging at 5000rpm for 3min, discarding most of supernatant, leaving about 100 ul, re-suspending thallus, and plating. And (3) carrying out inverted culture in an incubator at 37 ℃ overnight, picking clones on the plate for inoculation, extracting plasmid DNA, and carrying out PCR identification. Clones with the exact sequence were used for protein expression.

(2) Small amount of expression

Single colonies were picked from the transformed plates into 1.5ml of LB liquid medium, cultured at 37 ℃ and 200 rpm. Culturing until OD is 0.6-0.8, inducing with IPTG (0.5mM), and culturing at 37 deg.C and 200rpm for 2 h. Taking 1ml of induced bacteria liquid, centrifuging at 12000rpm for 1min, removing supernatant, blowing off the precipitate by 50-100 μ l of 10mM Tris-HCl (pH8.0) solution (the amount of the added buffer solution depends on the amount of the bacteria), adding 2 × Loading buffer with the same volume as the buffer solution, boiling for 5min at 100 ℃, and carrying out electrophoresis detection, wherein the electrophoresis result of the small-amount expressed protein is shown in figure 2.

(3) Large scale expression and purification

1-2. mu.l of the activated bacterial suspension was transferred to 5ml of LB liquid medium and cultured at 37 ℃ and 200 rpm. The cultured bacterial suspension was transferred to 500ml of LB liquid medium, mixed, cultured at 37 ℃ and 200rpm until the OD was about 0.8, and induced with IPTG (0.5mM) for 4 hours. Collecting bacteria with 400ml centrifuge tube, 6000rpm, centrifuging for 5 min. The supernatant was discarded. The pellet is blown off by 20-30ml 10mM Tris-HCl (pH8.0), 100 mul of bacterial suspension after ultrasonic treatment (the parameters: 500W power, 30 times of ultrasonic treatment, 10s each time, and 15s interval) is taken, 12000rpm is carried out, 10min is carried out by centrifugation, 50 mul of supernatant is taken to another centrifuge tube, after the supernatant is removed, the pellet is blown off by 50 mul of 10mM Tris-HCl (pH8.0) solution, 50 mul of 2 × loading buffer is added, the mixture is boiled for 5min at 100 ℃, and the expression condition is determined by electrophoresis. For bulk purification, the column was washed with purified water to pH 7.0, the nickel column was equilibrated with about 100ml of 10mM Tris-HCl (pH8.0) solution, and the sample was diluted to a final concentration of 0.5M sodium chloride in the sample by equilibrating the nickel column with about 50M of 10mM Tris-HCl (pH8.0) solution containing 0.5M sodium chloride. After the loading was completed, the column was washed with 10mM Tris-HCl (pH8.0) solution containing 0.5M sodium chloride. Then eluting with 10mM Tris-HCl (pH8.0) (containing 0.5M sodium chloride) solution containing 15mM imidazole, 60mM imidazole and 500mM imidazole, respectively, collecting protein peaks, electrophoretically analyzing the purified products, and storing the purified E-cadherin-F protein at-20 ℃ in the attached figure 3.

EXAMPLE 2 establishment of hybridoma cell lines

Immunization

The recombinant protein E-cadherin-F obtained in example 1 was emulsified with Freund's complete adjuvant (Sigma Co.), 6-week-old female ICR mice (purchased from Beijing Wahuafukang Biotech Co., Ltd.) were immunized, and 6 spots of each mouse were injected ventrally subcutaneously 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.) 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 suspension of spleen cells of a mouse which has reached the immune standard is prepared aseptically, mixed with mouse myeloma cells sp2/0(ATCC) at a ratio of 5:1, centrifuged at 1500rpm for 5 min. Abandoning the supernatant, putting the centrifuge tube into a water bath with the temperature of 37 DEG CIn (1), 1ml of PEG1500(Roche Co.) was slowly added 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 by picking

The size and density of the clone cell mass are moderate 7 days after fusion, the round, solid and large clone mass is absorbed under a dissecting mirror and is injected into a 96-hole culture plate which is prepared with a culture medium in advance, and 5 percent CO at 37 ℃ is put in₂Culturing in an incubator.

Fourth, ELISA screening positive hybridoma cell

After 3 days, the cell mass was about 2/3 basal areas, and 100. mu.l of the supernatant was screened by ELISA using recombinant protein and tumor cells, 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 was added to each well and incubated at 37 ℃ 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 an IgG2b subtype murine monoclonal antibody.

Second, determination of affinity constant

The recombinant protein of the E-cadherin fragment prepared in example 1 was taken 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 4 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 by using the following formulaThe affinity constant of the antibody is 5.07 x 10⁸。

Third, detecting E-cadherin protein by utilizing immunoblotting method

The recombinant E-cadherin-F protein was diluted to 2. mu.g/ml, loaded in 10. mu.l/well and subjected to 12% polyacrylamide gel electrophoresis. 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 a 5% skim milk powder in TBS-T (10mmol/L, Tris 0.9% NaCl in 1N HC1 adjusted to pH7.4 and Tween 20 added to a final concentration of 0.05%) blocking solution at 4 ℃ overnight. Monoclonal antibody 36C4 (1: 1000 dilution) was added at 1mg/ml and incubated overnight at 4 ℃. After washing the membrane with TBS-T buffer, add 1: a5000-diluted goat anti-mouse secondary antibody (Beijing Zhonghua Jinqiao Biotech Co., Ltd.) was incubated at room temperature for 1 hour. And washing the membrane with TBS-T, adding ECL (Beijing prilley Gene technology Co., Ltd.), and collecting chemiluminescence image data with ChemiDocMP multicolor fluorescence imaging system (Bio-Rad), wherein the detection result of the E-cadherin protein immunoblot is shown in figure 4, and the band signal in the result is clear, specific and single.

Example 5 determination of variable region sequences of antibodies

Culturing fresh hybridoma cells, taking the supernatant, verifying the antigen binding property, and centrifuging to collect 10⁶The hybridoma cells described above. Extracting total RNA of hybridoma cells by Trizol method. Mu.l of total RNA was taken, added with 2.5. mu.l of oligo (dT) 12-18 primer (10mM), and 5. mu.l of NTPs, mixed well, incubated at 70 ℃ for 5 minutes and then placed on ice for 5 minutes, or subjected to denaturation operation according to the reverse transcriptase used. Then, 5. mu.l of 5 Xreverse transcriptase buffer, 2.5. mu.l of DTT (0.1M) and 1. mu.l of reverse transcriptase (Beijing Huada protein research and development center, Ltd.) were added and reacted at 42 ℃ for 1 hour. The reaction was terminated by incubation at 70 ℃ for 15 minutes, and the obtained cDNA was stored at-20 ℃. The obtained first strand cDNA was subjected to PCR amplification, and 25pmol each of primers was added to a 50. mu.l reaction system, and the sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were in accordance withHippocampus, Inc., in the book "recombinant antibodies" (science publishers, 2005 publications) by Hippocampus, the design and synthesis of murine monoclonal antibody primer sequences (Biotechnology engineering (Shanghai) Co., Ltd.). The primers used for amplifying heavy chain variable regions were as follows, wherein 11 primers of mhv.b1 up to mhv.b12 were upstream primers, which can be used in combination with heavy chain downstream primer mhc.f, respectively, for amplifying heavy chain variable region genes.

MHV.B1:5’-GATGTGAAGCTTCAGGAGTC-3’

MHV.B2:5’-CAGGTGCAGCTGAAGGAGTC-3’

MHV.B3:5’-CAGGTGCAGCTGAAGCAGTC-3’

MHV.B4:5’-AGGTTACTCTGAAAGAGTC-3’

MHV.B5:5’-GAGGTCCAGCTGCAACAATCT-3’

MHV.B6:5’-GAGGTCCAGCTGCAGCAGTC-3’

MHV.B7:5’-CAGGTCCAACTGCAGCAGCCT-3’

MHV.B8:5’-GAGGTGAAGCTGGTGGAGTC-3’

MHV.B9:5’-GAGGTGAAGCTGGTGGAATC-3’

MHV.B10:5’-GATGTGAACTTGGAAGTGTC-3’

MHV.B12:5’-GAGGTGCAGCTGGAGGAGTC-3’

MHC.F:5’-GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3’

The primers used for amplifying the variable region of the light chain are as follows, wherein 10 primers from MKV.B1 to MKV.B10 are upstream primers which can be respectively combined with a light chain downstream primer MKC.F to amplify the variable region gene of the Kappa light chain.

MKV.B1:5’-GATGTTTTGATGACCCAAACT-3’

MKV.B2:5’-GATATTGTGATGACGCAGGCT-3’

MKV.B3:5’-GATATTGTGATAACCCAG-3’

MKV.B4:5’-GACATTGTGCTGACCCAATCT-3’

MKV.B5:5’-GACATTGTGATGACCCAGTCT-3’

MKV.B6:5’-GATATTGTGCTAACTCAGTCT-3’

MKV.B7:5’-GATATCCAGATGACACAGACT-3’

MKV.B8:5’-GACATCCAGCTGACTCAGTCT-3’

MKV.B9:5’-CAAATTGTTCTCACCCAGTCT-3’

MKV.B10:5’-GACATTCTGATGACCCAGTCT-3’

MKC.F:5’-GGATACAGTTGGTGCAGCATC-3’

The remaining dNTPs and buffer were added as usual, and finally 1. mu.l of cDNA template and 1U of hot-start Taq DNA polymerase (TaKaRa) were added. Setting PCR amplification program as 94 deg.c for 40 sec, 52 deg.c for 40 sec, 72 deg.c for 40 sec, 20-25 cycles, final extension at 72 deg.c for 3min, and setting the product at 4 deg.c for use or direct electrophoresis. 20 μ l of PCR product was analyzed by electrophoresis, separated on 1.5% agarose gel, recovered by cutting, and cloned into T-vector for sequencing.

Example 6 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 3min with PBS. Dropwise adding 3% H₂O₂Incubate for 10min and wash with PBS for 3 × 3 min. Spin-drying, slicing, and drippingThe released primary antibody (the dilution ratio of the antibody is designed according to the concentration of the antibody in the first dilution) is incubated for 1 hour at room temperature (25 ℃), PBS is washed for 3 multiplied by 3 minutes, secondary antibody is added dropwise and incubated for 15 to 30 minutes at room temperature, PBS is washed for 3 multiplied by 3 minutes, PBS is thrown off, and DAB developing solution which is freshly prepared is used for developing for 3 to 10 minutes. 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 3min, 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 (36C4) and the commercial antibody (the murine monoclonal antibody NCH-38) are synchronously detected on different human tumor tissue chips (including cervical squamous carcinoma, ductal carcinoma of mammary gland and the like) and the detection results are compared.

And (5) carrying out statistics on the immunohistochemical result of the E-Cadherin. The whole experimental process adopts a double-blind design, and the statistical results are shown in the following table.

The positive coincidence rate of the antibody (36C4) and the commercial antibody (murine monoclonal antibody NCH-38) was 100%, the negative coincidence rate was 100%, and the total coincidence rate was 99%, and in 10 tumor tissue samples, the staining intensity of the antibody was higher than that of the commercial antibody, indicating that the specificity of the antibody in tumor tissues was equivalent to that of the commercial antibody, and the sensitivity was higher than that of the commercial antibody.

The specific dyeing results are as follows:

FIG. 5 is a graph comparing the staining results of breast cancer sample 1; the staining results were +++, for 36C4 and ++, for NCH-38.

FIG. 6 is a graph comparing the staining results of breast cancer sample 2; the staining results were ++, for 36C4 and + for NCH-38.

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 comprises 3 different tissue samples, each derived from a different person. 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 (36C4) and the commercial antibody (murine monoclonal antibody NCH-38) were detected simultaneously on a normal tissue chip, and the detection results were completely consistent, indicating that the specificity of the antibody in normal tissue was equivalent to that of the commercial antibody.

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.

Sequence listing

<110> Fuzhou mai New Biotechnology development Co., Ltd

<120> monoclonal antibody of anti-E-cadherin protein, cell strain, preparation method and application thereof

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Claims (7)

1. An anti-E-cadherin monoclonal antibody is produced by a hybridoma cell strain with the preservation number of CGMCC NO 15489.

2. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes an E-cadherin protein.

3. The monoclonal antibody according to claim 2, which specifically recognizes a protein fragment represented by SEQ ID No.1 in the E-cadherin protein.

4. 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.

5. The monoclonal antibody according to claim 1, 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.

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

7. A hybridoma cell strain secreting E-cadherin-resistant protein molecules is a mouse hybridoma cell strain 36C4, and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO 15489.

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