CN108467432A

CN108467432A - The monoclonal antibody and its cell strain, preparation method and application of anti-E-cadherin albumen

Info

Publication number: CN108467432A
Application number: CN201810539838.6A
Authority: CN
Inventors: 陈滢; 杨清海; 周洪辉; 高惠然; 王小亚
Original assignee: Fuzhou Maixin Biotechnology Development Co ltd
Current assignee: Fuzhou Maixin Biotechnology Development Co ltd
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2018-08-31
Anticipated expiration: 2038-05-30
Also published as: CN108467432B

Abstract

The present invention relates to the purposes of the preparation method and the monoclonal antibody of the monoclonal antibody that can detect E cadherin and its hybridoma cell strain 36C4.The antigen for preparing the antibody is one section and is selected from E cadherin protein recombinant proteins, mouse is immunized through Bacillus coli expression and after purification, and with the screening of recombinant protein and tumour cell progress hybridoma cell strain, the antibody finally obtained belongs to IgG2b hypotypes.After the 36C4 cell strains extraction RNA obtained to screening, the reverse transcription and cDNA to obtain is template expands the sequence of its encoding antibody variable.The antibody of the present invention can be used for immune detection.

Description

The monoclonal antibody and its cell strain of anti-E-cadherin albumen, preparation method and Using

Technical field

The present invention relates to biomedical engineering field, more particularly to a kind of anti-E-cadherin protein monoclonal antibodies and its Cell strain, preparation method and application.

Background technology

E-Cadherin is the cell adhesion protein (Ca that calcium ion relies on²⁺dependent cell adhesion Molecule family) one of family member, also referred to as mulberry body attachment proteins (Uvomorulin), L-CAM or Cell- CAM120/80.There is another two protein member, N-Cadherin and P-Cadherin in the family.Different attachment proteins molecules Respectively there are unique Tissue distribution form, expression different with cell growth, developmental condition difference in vivo.E-Cadheri is one Kind can trigger the upper hide collagen of the cross-film of intercellular adhesion, and missing is related with tumour diffusion, and function is different in tumour cell It may cause cell detachment when often and shift.The albumen is generally film dyeing and cytoplasm dyeing.This albumen is in diffusion Expression declines in carcinoma of urinary bladder, and the tumour of breast portion is different, nothing in lobular carcinoma and lobular carcinoma in situ of breast (LCIS) It expresses, being expressed in duct adenocarcinoma reduces, and is positive in small cell carcinoma and carcinoma in situ (DCIS).The signet ring cell cancer of stomach Without expression in (signet-ring cell carcinoma), in adenocarcinoma of colon, compared with the epithelial cell to misplace in adenoma, The reduction of film dyeing can occur.

In histopathology, the mark of mammary gland infiltration cancer is the missing of musculoepithelia cell layer around cancer stove.Immunohistochemistry is The important supplementary means of mammary gland early invasive carcinoma pathological diagnosis, common musculoepithelia cell layer marker are smooth myosin weight Chain (SMM-HC), actin binding protein (Calponin) and p63.However, the marker of single myoepithelium can not Determine whether a small number of microcell groups for lacking musculoepithelia cell layer are cancer cell group in interstitial.Seedling is numerous to be used E- (the numerous of seedling exempts from the diagnostic level that Cadherin antibody improves mammary gland early invasive carcinoma with the double methods contaminated of Calponin antibody Application Zhengzhou University Master's thesis of the double dye methods of epidemic disease groupization in mammary gland early invasive carcinoma pathological diagnosis, 2016.)

Use to E-cadherin associated antibodies, detected with immunohistochemistry based on, but also have with ELISA or antibody core The detection method of sheet mode, a kind of patent of invention " antibody chip that can detect various kinds of cell adhesion factor simultaneously " (publication number CN107328941A a kind of antibody chip that can detect various kinds of cell adhesion factor simultaneously) is disclosed, wherein also including E- The detection of cadherin, the E-cadherin albumen inorganization and cellular localization information of this method detection are not suitable for most of Pathologic diagnosis of tumor is used.In addition, the antibody can be additionally used in the enrichment and purifying of Iisolated tumor cells in the circulatory system, such as invent Patent " a kind of E- cadherin, the multiple antibody immune magnetic beads of Cadherin-11, EpCAM and preparation method thereof " (publication number CN106432504A) the enrichment i.e. in the method for magnetic bead coupling by multiple antibody for CTC cells in blood, likewise, this is anti- Body does not have the detection applications of immunohistochemistry pathology.In patent of invention " the anti-E-Cadherin albumen for turning to purposes with immune group The anti-E-Cadherin monoclonal antibodies and application of monoclonal antibody hybridoma cell and its generation " (application number： 201510703037.5) a kind of utilize is described in has transfected the HEK293 cells of E-cadherin genes as immunogene system The method of standby monoclonal antibody, the antibody can be used for immunohistochemistry and immune-blotting method, and immunogene is the gene of overall length, More than the 30 of Cadherin families in a member, similarity is very high, so it identifies that specificity need to adequately be confirmed.

Visible a variety of scientific research class commercial antibodies in the market.But for immunohistochemistry detection when specificity and susceptibility and Coloration result is not enough to breath.The companies such as Zymed, Thermo Fisher mouse monoclonal antibody 4A2C7, Leica on sale The rabbit monoclonal antibody product HPA004812 of the 36B5 and Sigma-Aldrich (MERCK) of Microsystems companies can be used for being immunized Histochemical analysis, but effect is different, may continue to improve on susceptibility and specificity.

Invention content

A kind of anti-E-cadherin monoclonal antibodies are inventor provided, are the miscellaneous of CGMCC NO 15489 by preserving number Tumor cell strain is handed over to generate.It is general that the cell strain has been preserved in China Committee for Culture Collection of Microorganisms on March 9th, 2018 Logical microorganism center, Classification And Nomenclature are：Mouse hybridoma cell system, preserving number are：CGMCC NO 15489, address are Beijing No. 3 Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1.

Further, the monoclonal antibody specificity identifies E-cadherin albumen.

Further, amino acid shown in the SEQID1 in the monoclonal antibody specificity identification E-cadherin albumen Sequence.

Further, the DNA sequence dna of the heavy chain variable region of the monoclonal antibody is nucleotides sequence shown in SEQ ID3 The DNA sequence dna of row, the light chain variable region of the monoclonal antibody is nucleotide sequence shown in SEQ ID4.

Further, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is amino acid shown in SEQ ID5 Sequence；The amino acid sequence of the monoclonal antibody light chain variable region is amino acid sequence shown in SEQ ID6.

Further, the monoclonal antibody is mouse IgG 2a hypotype monoclonal antibodies.

Inventor additionally provides a kind of preparation method of anti-E-cadherin monoclonal antibodies, chooses E-cadherin eggs White the 498th as immunogene and carries out Protein reconstitution to 709 amino acids, and recombinant protein recombinates table via Escherichia coli It reaches.The both sides of immunogene are merged respectively has 6 histidines as label.

Inventor additionally provides the hybridoma cell strain of one plant of anti-E-cadherin protein molecular of secretion, and the cell strain is Mouse hybridoma cell strain 36C4, the cell strain have been preserved in Chinese microorganism strain preservation management committee on March 9th, 2018 Member's meeting common micro-organisms center, Classification And Nomenclature are：Mouse hybridoma cell system, preserving number are：CGMCC NO 15489, address are The Institute of Microorganism, Academia Sinica of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

Inventor additionally provides purposes of the said monoclonal antibody in the detection of E-cadherin protein immunizations.

Further, the immune detection includes immunohistochemical method, Western blot and enzyme-linked immunization.

It is different from the prior art, advantageous effects are caused by the present invention：

(1) monoclonal antibody that obtains of the present invention is generated by hybridoma 36C4 secretions, can specific recognition recombinant expression and E-cadherin albumen in human epithelial cells can detect normal structure epithelium, galactophore epithelial cell, wing with specific detection The expression of E-cadherin albumen in Guang cancerous tissue.

(2) the hybridoma 36C4 that the present invention obtains is a kind of Ig2b subclass antibodies, the knot with natural E-cadherin albumen Conjunction has extremely strong specificity and sensibility.

(3) because the monoclonal antibody hybridoma 36C4 of the invention obtained is with extracellular membrane-proximal region segment, optimized codon sequence Synthetic gene segment has good surface accessibility, suitable secondary structure and ammonia as immunogene, the region after row Base acid composition characteristic, the antibody of preparation can meet immunohistochemistry (IHC), immunoblotting analysis (Western blotting) with And the testing requirements of ELISA method.

Description of the drawings

Fig. 1 is the front and back comparison chart of YE-cadherin gene fragment orders optimization；1 is the sequence E- after optimization Cadherin-F, 2 be the partial sequence of original reference gene order NM_004360.4, and the two similarity is 76.5%, altogether to it In 150 bases be optimized.

Fig. 2 is the electrophoresis result figure expressed in a small amount for recombinating E-cadherin albumen；M：Molecular weight marker, 1-5：At random 5 monoclonal bacterial strains of picking；

Fig. 3：The electrophoresis result figure of the E-cadherin-F albumen of purifying；M：Molecular weight marker, E-cadherin-F：Through miaow The recombination E-cadherin-F albumen of azoles elution；

Immunoblotting of the monoclonal antibody of Fig. 4 purifying to E-cadherin albumen；E-cadherin albumen concentration is recombinated in experiment For 2 μ g/ml, 10 μ l of electrophoresis loading, total amount 20ng, 67127-36C4 antibody concentration is 1mg/ml, 1:1000 dilutions use, HRP enzyme marks sheep anti mouse 1:5000 use.

Fig. 5 is the coloration result comparison diagram of breast cancer sample 1；

Fig. 6 is the coloration result comparison diagram of breast cancer sample 2.

Specific implementation mode

For the technology contents of technical solution, construction feature, the objects and the effects are described in detail, below in conjunction with specific reality It applies example and attached drawing is coordinated to be explained in detail.

It is prepared by 1 recombinant protein of embodiment：

From Uniprot databases (http://www.uniprot.org) in select number for P12830 E- For cadherin protein sequences as standard sequence, sequence passes through BLAST (https as shown in sequence table ID1:// Blast.ncbi.nlm.nih.gov/Blast.cgi) tool compares its sequence difference with other with family protein, and with The Protean modules of DNASTAR8.0 softwares (www.dnastar.com) carry out secondary structure, antigenicity and surface accessibility point Analysis.For the 498th of selected E-cadherin albumen to 709 amino acids as recombinant protein, which closes on the born of the same parents of transmembrane region Outskirt.

(1) gene chemical synthesis and clone

To improve the yield of expression, the optimization of codon is carried out to the DNA of selection area, nucleic acid fragment is through gene chemical synthesis Mode obtains (Sangon Biotech (Shanghai) Co., Ltd.), for ease of clone, in the upstream and downstream point of coded sequence BamHI and XhoI restriction enzyme sites are not added, segment is recycled after BamHI and XhoI double digestions, are connected into through same double digestion Expression vector pET30a (Novagen companies are now Merck companies) is linearized, the genetic fragment name of expression vector is cloned into For E-cadherin-F.

It includes the removal of restriction enzyme site, coding region G/C content that the factor considered is needed in codon optimisation process Adjustment, Escherichia coli rare codon replacement and low preference codon be adjusted to host's high preference codon, password The optimization of son can improve yield, the stability of transcription product, and the expression of protein can be improved.E-cadherin albumen Reference sequences in Genbank are NM_004360.4, and the present invention carries out sequence optimisation in this sequence basis.Optimization is commented Valence index is codon adaptability index (CAI), and the index is better closer to 1, and sequence G/C content should be controlled 30% to 70% Between, the lower the series connection degree (CFD) of rare codon should be the better less than 30%.After sequence optimisation, the codon of aim sequence Adaptation index is increased to 0.87 by 0.61, and G/C content is adjusted to 50.44%, CFD values by 48.74% and is reduced to 0 by 10%.Sequence The front and back comparison result of optimization is shown in attached drawing 1, the front and back comparison chart of YE-cadherin gene fragment orders optimization.The synthetic gene Expression yield be higher than general gene, also indicate that sequence optimisation achieves advantageous effect.

Concrete scheme is as follows：

Genetic fragment double digestion

37 DEG C of digestion 3h, digestion products electrophoresis detection recycle the DNA fragmentation of digestion for connecting.

37 DEG C of digestion 3h, digestion products electrophoresis detection recycle the pET30a carrier segments of linearisation for connecting.

Connection and conversion：

Linearize 3 μ l of pET30a1 carriers

2 μ l of endonuclease bamhi

Connect enzyme mixation (TaKaRa) 5 μ l

Mixing, 16 DEG C connection overnight, take out -80 DEG C preservation e. coli bl21 competent cells (Novagen companies, It is now Merck companies), it is placed on ice slowly defrosting, connection product is added, 800 μ l are added in 42 DEG C of heat shock 90s after ice bath 30min The LB culture mediums of non-resistant, 37 DEG C of culture 45min, 5000rpm centrifugation 3min, abandon most of supernatant, stay about 100 μ l, bacterium is resuspended Body, coated plate.Overnight incubation is inverted in 37 DEG C of incubators, clone's inoculation on picking tablet extracts Plasmid DNA, carries out PCR mirror It is fixed.The right-on clone of sequence is used for protein expression.

(2) a small amount of expression

From picking monoclonal to 1.5ml LB liquid mediums in the tablet of conversion, 37 DEG C, 200rpm cultures.Culture It is induced to OD=0.6-0.8, IPTG (0.5mM), 37 DEG C, 200rpm cultivates 2h.The bacterium solution for taking 1ml to induce, 12000rpm, centrifugation 1min, abandons supernatant, and precipitation is dispelled with 50-100 μ l 10 mM Tris-HCl (pH8.0) solution and (amount of buffer solution is added regarding thalline Depending on amount), the 2 × Loading buffer isometric with buffer solution is added, 100 DEG C are boiled 5min, and electrophoresis detection is expressed in a small amount Protein electrophoresis result is shown in attached drawing 2.

(3) great expression and purifying

The bacterium solution of 1-2 μ l activation is transferred in 5ml LB liquid mediums, 37 DEG C, 200rpm, culture.By the bacterium of culture Liquid is transferred to the mixing of 500ml LB liquid mediums, and 37 DEG C, 200rpm, culture to OD is about 0.8, and IPTG (0.5mM) is added and lures Lead 4h.Bacterium is received with 400ml concentrator bowls, 6000 rpm centrifuge 5min.Abandon supernatant.Precipitation 20-30ml 10mM Tris-HCl (pH 8.0) solution dispels, and takes the ultrasonic (parameters of 100 μ l：Power 500W, 30 times, each ultrasound 10s is spaced 15s) after bacterium hang Liquid, 12000rpm centrifuge 10min, take 50 μ l supernatants to another centrifuge tube, 50 μ l 10mM of precipitation after supernatant removal is clean Tris-HCl (pH 8.0) solution dispels, and 50 μ 2 × loading of l buffer is added, 100 DEG C are boiled 5min, and electrophoresis determines expression Situation.When large-scale purification, column is washed to pH 7.0, with 10mM Tris-HCl (pH 8.0) solution equilibria nickel of about 100ml with pure water Column, then l sodium chloride containing 0.5M with about 50m 10mM Tris-HCl (pH 8.0) solution equilibria nickel column, dilute sample loading, Final concentration of 0.5M sodium chloride-containings in sample.It is molten with the 10mM Tris-HCl (pH 8.0) of the sodium chloride containing 0.5M after end of the sample Liquid washes column.(contain 0.5M with the 10mM Tris-HCl (pH 8.0) containing 15 mM imidazoles, 60mM imidazoles, 500mM imidazoles respectively later Sodium chloride) solution elution, collect protein peak respectively, electrophoretic analysis purified product, it is purified after E-cadherin-F albumen electricity Swimming result is shown in that attached drawing 3, -20 DEG C of albumen sample save backup.

The foundation of 2 hybridoma cell line of embodiment

One, it is immunized

The recombinant protein E-cadherin-F obtained in embodiment 1 is emulsified with Freund's complete adjuvant (Sigma companies), is exempted from 6 week old Female ICR mice of epidemic disease (is purchased from Fukang bio tech ltd of Beijing China), 6 points of every mouse of abdominal part hypodermic, agent Amount is 60 μ g/.Every 14 days booster immunizations are primary, and using Freund, non-fully adjuvant (Sigma companies) emulsifies antigen, dosage 30 μ g/ are only.7 days how anti-effects that mice serum moderate resistance immunogene is detected with indirect ELISA (wavelength 450nm) after 3rd booster immunization Valence, the highest mouse of potency is immune with tail vein injection impact, antigen physiological saline mixing, and dosage is 50 μ g/.

Two, cell fusion

It is sterile to prepare immune mouse boosting cell suspension up to standard, with murine myeloma cell sp2/0 (ATCC) with 5:1 ratio Example mixing, centrifuges 1500rpm, 5min.Centrifuge tube is put into 37 DEG C of water-baths after abandoning supernatant, is slowly added to 1ml's in 1 minute PEG1500 (Roche companies), and stir cell.After standing 1min in warm water, IMDM (the Sigma public affairs of 10ml serum-frees are added Department), mixing centrifuges 1000rpm, 5min.After abandoning supernatant, addition 10ml serum (PAA companies) is careful to blow and beat cell Come, and the thymocyte of 5ml mixing 10xHAT (Sigma companies), mixing is added.It adds 25ml and contains 2.1% nitrocellulose The semisolid culturemedium of plain (Sigma companies) mixes well, and then uniformly pours into 20 Tissue Culture Dish.By cell culture Ware is put into wet box, and 37 DEG C of 5%CO are put into₂It is cultivated in incubator.

Three, clone is chosen

7 days clone cells roll into a ball size medium density after fusion, under anatomical lens, draw round, real, big cloning cluster and squeeze into thing It is first ready in 96 well culture plates of culture medium, is put into 37 DEG C of 5%CO₂It is cultivated in incubator.

Four, ELISA screens positive hybridoma cell

After 3 days, cell concentration accounts about floor space 2/3, and 100 μ l supernatants recombinant proteins and tumour cell is taken to carry out respectively ELISA is screened.Positive colony changes liquid completely, and the complete culture that 200 μ l contain feeder cells and 1%HT (Sigma companies) is added Base.Second of ELISA screening is carried out two days later, and positive colony is transferred to gets out the 24 of culture medium (containing feeder cells and HT) in advance Orifice plate culture.100 μ l supernatants are taken to carry out third time ELISA screenings after five days, positive colony is gradually transferred to 6 orifice plates and cell culture Bottle, which expands, to be cultivated and freezes.

3 ascites of embodiment induces method and prepares monoclonal antibody

One, prepared by ascites

Exponential phase cell is washed and has been hanged with serum free medium, counts about 5 × 10⁵, 1ml.The cell abdomen of suspension The mouse of paraffin oil sensitization is used in chamber injection in advance.Start to collect ascites after 7 days.The ascites of taking-up centrifuges 4000rpm in 4 DEG C, 10min.The intermediate ascites of careful suction is collected in centrifuge tube, 4 DEG C or -20 DEG C preservations.

Two, the purifying of monoclonal antibody

With HiTrap rProtein A FF (GE companies) affinity chromatography by specifications antibody purification from ascites.SDS- PAGE glue identifies purity, Bradford method measured concentrations.The antibody of purifying is stored in -20 DEG C.

4 monoclonal antibody CHARACTERISTICS IDENTIFICATION of embodiment

One, subgroup identification

Coating sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge) is diluted with 100mM PBS (pH7.4) extremely 0.5 μ g/ml add 100 μ l per hole, 4 DEG C, stay overnight.It is emptied liquid, is washed 3 times with the PBS (PBS-T) containing 0.05%Tween, per hole 200 μ l confining liquids (PBS containing 2%BSA and 3% sucrose), 37 DEG C of incubation 1h are added.It is emptied liquid, is cleaned 3 times with PBS-T. 0.1ml hybridoma supematants, 37 DEG C of incubation 1h are added per hole.Liquid is emptied to be cleaned 3 times with PBS-T.With confining liquid 1：1000 dilutions Sheep anti mouse (κ, the λ) antibody or 1 of HRP labels：2000 dilution HRP label sheep anti mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech companies) 0.1ml is separately added into hole appropriate per hole, 37 DEG C of incubation 1h.Incline Empty liquid is cleaned 3 times with PBS-T.50 μ l are added to contain 0.15% ABTS (Southern Biotech companies) and 0.03% per hole H₂O₂Citrate buffer solution (PH4.0) carry out chromogenic reaction, the OD values under 405nm wavelength are measured in 10-20min.

The results show that monoclonal antibody of the present invention is IgG2b hypotype mouse resource monoclonal antibodies.

Two, affinity costant measures

The E-cadherin segment recombinant proteins prepared in Example 1, peridium concentration be 2 μ g/ml, 100 holes μ l/, 4 DEG C coating overnight, PBS-T is washed 3 times.37 DEG C of 200 μ l confining liquids are added to close 2h per hole, PBS-T is washed 3 times.It is purified in embodiment 4 Monoclonal antibody, from 1：200 start 2 times of gradient dilutions, and last 1 hole blanks control, and 37 DEG C are incubated 1h, and PBS-T is washed 3 times.HRP The sheep anti mouse secondary antibody 1 of label：20000 dilutions, per 100 μ l of hole, 37 DEG C are incubated 1h, and PBS-T is washed 3 times.100 μ l are added per hole to contain 0.1% TMB (Sigma companies) and 0.03%H₂O₂Citrate phosphate buffer develop the color 10min, add 50 μ l0.5M sulfuric acid molten Liquid terminates reaction.The light absorption value of wavelength 450nm is measured with microplate reader.The curve that OD values correspond to antibody extension rate is drawn, is found Corresponding extension rate A when the half of maximum combined OD values, the affinity costant that the antibody is calculated using following equation are 5.07 ×10⁸。

Three, E-cadherin albumen is detected using Western blot

The E-cadherin-F albumen of recombination is diluted to 2 μ g/ml, per 10 μ l of hole loading, carries out 12% polyacrylamide Gel electrophoresis.Gel protein band is transferred on pvdf membrane (Millipore in Bio-Rad electrotransfer systems according to a conventional method Company).Film is placed in the TBS-T containing 5% skimmed milk power, and (10mmol/L, Tris contain 0.9%NaCl, extremely with 1N HC1 tune pH 7.4, final concentration of 0.05% polysorbas20 being added) 4 DEG C overnight in confining liquid.The monoclonal antibody 36C4 (1 of 1mg/ml is added： 1000 dilutions), 4 DEG C of overnight incubations.After washing film with TBS-T buffer solutions, it is added 1：5000 diluted sheep anti mouse secondary antibodies (China fir in Beijing Bioisystech Co., Ltd of Golden Bridge), it is incubated at room temperature 1 hour.Film is washed with TBS-T again, the super quick developing solutions of ECL are added, and (Beijing is general Sharp lema gene Technology Co., Ltd.), chemiluminescence image number is carried out with ChemiDocMP multicolor fluorescences imaging system (Bio-Rad) According to acquisition, E-cadherin protein immunoblot testing results are shown in attached drawing 4, as a result in Band signal it is clear, special, it is single.

The variable region sequences of 5 antibody of embodiment measure

Fresh hybridoma is cultivated, after taking supernatant to carry out antigenic binding property verification, is collected by centrifugation 10⁶The above hybridization Oncocyte.Trizol methods extract hybridoma total serum IgE,.9 μ l total serum IgEs are taken, 2.5 μ Loligo (dT), 12-18primer are added (10mM) and 5 μ ldNTPs, be uniformly mixed, 70 DEG C of heat preservations are set 5 minutes on ice after five minutes, or according to the reverse transcriptase used into Row denaturation operation.5 × reverse transcriptase buffer of 5 μ l, 2.5 μ l DTT (0.1M) and 1 μ l reverse transcriptases (Beijing is then added Co., Ltd of Hua Da protein research and development centre), 42 DEG C are reacted 1 hour.70 DEG C are incubated 15 minutes to terminate reaction, the cDNA of acquisition It is stored in -20 DEG C.First chain cDNA of acquisition is subjected to PCR amplification, each 25pmol of primer is added in 50 μ l reaction systems, Heavy chain variable region and the sequence of light chain variable region amplification primers put forth energy chief editor's according to Shen again《Recombinant antibodies》(Science Press, Publish within 2005) design of mouse monoclonal antibody primer sequence and synthesis (Sangon Biotech (Shanghai) Co., Ltd.) in a book.With In amplification heavy chain variable region primer it is as follows, wherein MHV.B1 until MHV.B12 11 primers be sense primer, can respectively with The MHC.F combinations of heavy chain downstream primer are for expanding heavy chain variable region gene.

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’

Primer for expanding light chain variable region is as follows, and wherein MKV.B1 is until 10 primers of MKV.B10 draw for upstream Object can combine the variable region gene for expanding Kappa light chains with light chain downstream primer MKC.F respectively.

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’

Remaining dNTPs and buffer solution are eventually adding 1 μ l and 1U thermal starting Taq DNA of cDNA templates according to being routinely added to Polymerase (TaKaRa).Be arranged PCR amplification program be 94 DEG C 40 seconds, 52 DEG C 40 seconds, 72 DEG C 40 seconds, carry out 20 to 25 cycle, Last 72 DEG C extend 3 minutes, and product can be placed in 4 DEG C of spare or direct electrophoresis.20 μ l PCR products are taken to carry out electrophoretic analysis, Gel extraction is detached on 1.5% Ago-Gel, is cloned into carrier T sequencing.

6. organization chip of embodiment dyes and identification

One, chip fabrication process

Row HE slices dyeing advanced to each sample, to determine tumor locus.Use the full-automatic tissue of 3DHISTECH companies Chip instrument makes organization chip.Manufactured organization chip wax stone is placed into wax stone and makes mold, is put into 68 DEG C of ovens 10 points Clock makes the wax of cured piece of tissue core and receptor combine together, mold is then gently taken out from oven, the paraffin of partly to melt state is allowed to exist Organization chip wax stone, is removed from the molds after placing into -20 DEG C of refrigerator freezing 6min, cuts by cooling about 30min under room temperature Piece or be put into 4 DEG C of refrigerators saves backup.Serial section is carried out after repairing piece, thickness is set to 3 μm, and serial section is floated on 40% wine In essence, it is allowed to be unfolded naturally, then separated slice is transferred in 50 DEG C of warm water and opens up piece 30 seconds, handled with through poly-D-lysine The glass slide mount slice crossed, manufactured organization chip is put into 68 DEG C of ovens and bakes piece 2 hours, is taken out, and room temperature cooling is put Enter -4 DEG C of refrigerators to preserve.

Two .IHC are dyed and analysis

Conventional xylene dewaxes 3 times, 6 minutes every time, aquation in 100%, 100%, 95%, 85% graded ethanol, and every time 3 Minute, last tap water rinses.Antigen retrieval is carried out, then slice is put into wet box, PBS is rinsed 3 × 3 minutes.It is added dropwise 3% H₂O₂It is incubated 10 minutes, PBS is rinsed 3 × 3 minutes.Drying slice, the diluted primary antibody of proper proportion is added dropwise, and (dilution is according to anti-for the first time Bulk concentration carrys out the dilution ratio of designerantibodies) incubation 1 hour of (25 DEG C) of room temperature, PBS is rinsed 3 × 3 minutes, and secondary antibody room temperature is added dropwise and incubates It educates 15-30 minutes, PBS is rinsed 3 × 3 minutes, gets rid of PBS, is developed the color 3-10 minutes with the DAB developing solutions of fresh configuration.Haematoxylin It redyes 25 seconds, PBS returns 30 seconds blue.According to 85% (3 minutes) -95% (3 minutes) -100% (3 minutes) -100% (3 minutes) Alcohol gradient is dehydrated successively, transparent 3 minutes of last dimethylbenzene, neutral gum mounting.

Immunohistochemical staining result is divided into：It is positive and negative.Positive expression must be in cell and the specific antigen portion of tissue Position can just be considered as the positive.It is distributed clearly in tissue staining and cellular localization is accurate, coloration result is according to staining power Difference further divided, it is specific as follows：

1, sample is weakly positive；Labeled as "+"；

2, sample is moderate positive；Labeled as " ++ "；

3, sample is High positive；Labeled as " +++ ".

4, sample is feminine gender, is labeled as "-".

Three, data statistics

1, tumor tissue array testing result：

By this antibody (36C4) and commercial antibody (mouse monoclonal antibody NCH-38) in different human body tumor tissue array (including uterine neck Squamous carcinoma, breast ductal cancer etc.) on synchronize detect and compare testing result.

The ImmunohistochemistryResults Results of E-Cadherin are counted.Entire experiment process takes double-dummy design, and statistical result is such as Following table.

The positive coincidence rate of this antibody (36C4) and commercial antibody (mouse monoclonal antibody NCH-38) is 100%, and negative match-rate is 100%, total coincidence rate is 99%, and in 10 tumor tissues samples, the staining power of this antibody is higher than the dyeing of commercial antibody Intensity illustrates that this antibody is suitable with commercial antibody in the specificity of tumor tissues, and susceptibility is more than commercial antibody.

Specific coloration result is visible：

Fig. 5 is the coloration result comparison diagram of breast cancer sample 1；The coloration result of 36C4 is +++, the dyeing knot of NCH-38 Fruit is ++.

Fig. 6 is the coloration result comparison diagram of breast cancer sample 2；The coloration result of 36C4 is ++, the coloration result of NCH-38 For+.

2, normal structure chip test result：

Normal structure chip includes 30 kinds of normal structure samples, and normal structure sample is mainly selected from fresh, fixed in time Specimens from pri；Each tissue includes 3 different tissue samples, and each tissue samples are respectively derived from different people.30 kinds just Often tissue includes：Brain, heart, cerebellum, oesophagus, adrenal gland, stomach, ovary, small intestine, pancreas, Colon and rectum, parathyroid gland, liver, Hypophysis, salivary gland, testis, kidney, thyroid gland, prostate, mammary gland, uterus, spleen, bladder, tonsillotome, skeletal muscle, thymus gland (child), Skin, marrow, peripheral nerve, lung, mesothelial cell.

This antibody (36C4) and commercial antibody (mouse monoclonal antibody NCH-38) are synchronized into detection on normal structure chip, examined Survey result is completely the same, illustrates that this antibody is suitable with commercial antibody in the specificity of normal structure.

It should be noted that herein, relational terms such as first and second and the like are used merely to a reality Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to Non-exclusive inclusion, so that process, method, article or terminal device including a series of elements include not only those Element, but also include other elements that are not explicitly listed, or further include for this process, method, article or end The intrinsic element of end equipment.In the absence of more restrictions, being limited by sentence " including ... " or " including ... " Element, it is not excluded that there is also other elements in process, method, article or the terminal device including the element.This Outside, herein, " being more than ", " being less than ", " being more than " etc. are interpreted as not including this number；" more than ", " following ", " within " etc. understandings It includes this number to be.

It should be noted that although the various embodiments described above have been described herein, it is not intended to limit The scope of patent protection of the present invention.Therefore, based on the present invention innovative idea, to embodiment described herein carry out change and repair Change, or using equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it directly or indirectly will be with Upper technical solution is used in other related technical areas, is included within the scope of patent protection of the present invention.

Sequence table

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<120>The monoclonal antibody and its cell strain, preparation method and application of anti-E-cadherin albumen

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Claims

1. a kind of anti-E-cadherin monoclonal antibodies are generated by the hybridoma cell strain that preserving number is CGMCC NO 15489.

2. monoclonal antibody according to claim 1, which is characterized in that the monoclonal antibody specificity identifies E- Cadherin albumen.

3. monoclonal antibody according to claim 2, which is characterized in that the monoclonal antibody specificity identifies E- Amino acid sequence shown in SEQID1 in cadherin albumen.

4. monoclonal antibody according to claim 1, which is characterized in that the heavy chain variable region of the monoclonal antibody DNA sequence dna is nucleotide sequence shown in SEQ ID2, and the DNA sequence dna of the light chain variable region of the monoclonal antibody is SEQ ID3 Shown in nucleotide sequence.

5. monoclonal antibody according to claim 1, which is characterized in that the ammonia of the heavy chain variable region of the monoclonal antibody Base acid sequence is amino acid sequence shown in SEQ ID4；The amino acid sequence of the monoclonal antibody light chain variable region is SEQ Amino acid sequence shown in ID5.

6. monoclonal antibody according to claim 1, which is characterized in that the monoclonal antibody is mouse IgG 2b hypotypes Monoclonal antibody.

7. a kind of preparation method of anti-E-cadherin monoclonal antibodies, it is characterised in that：Choose the of E-cadherin albumen 498 as immunogene and carry out Protein reconstitution to 709 amino acids, and recombinant protein is via Recombinant protein expression.

8. the hybridoma cell strain of one plant of anti-E-cadherin protein molecular of secretion, the cell strain is mouse hybridoma cell strain 36C4, the cell strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is： CGMCC NO 15489。

9. any monoclonal antibodies of claim 1-6, the purposes in the detection of E-cadherin protein immunizations.

10. immune detection according to claim 9, which is characterized in that the immune detection includes immunohistochemical method, Western blot and enzyme-linked immunization.