CN108084264A - Anti-human B7-H1 method for preparing monoclonal antibody and its immunohistochemistry detect apply - Google Patents
Anti-human B7-H1 method for preparing monoclonal antibody and its immunohistochemistry detect apply Download PDFInfo
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Abstract
The invention discloses a kind of anti-human B7 H1（PD‑L1）The detection application and the identification of heavy chain of antibody, light-chain variable sequence of monoclonal antibody and its immunohistochemistry, preparation and this plant of monoclonal antibody the present invention relates to anti-human B7 H1 monoclonal antibodies can be used for immunohistochemistry Samples detection and tumor tissues B7 H1 to analyze, and heavy chain variable region (mVH) and light chain variable region (mVL), sequence verification B7 H1 weight light chain variable sequences are extracted from the hybridoma for secreting anti-human B7 H1 monoclonal antibodies.Implement eucaryotic cell strain expression antibody on this basis, obtained antibody empirical tests have good binding ability, using Immunohistochemical detection to tumor specimen specific stain, and the effect and specificity that the B7 H1 monoclonal antibodies more voluntarily developed dye same tissue specimen with commercialization B7 H1 antibody.
Description
Technical field
Detection application and heavy chain of antibody variable region and light chain variable region sequence the present invention relates to a kind of antibody mediated immunity group
The verification of row, belongs to biological technical field, the preparation method of more particularly to anti-human B7-H1 monoclonal antibodies and its in immunohistochemistry
The application of detection.
Background technology
Tumor microenvironment is the critical positions of tumour cell and host immune system interaction.It understands in depth in microenvironment
The mechanism of immunocyte and tumour immunity is to improve body tumor immunity, establishes the pass of effective cancer immunotherapy means
Key.The microenvironment that tumour cell is formed with human immune system promotes tumor immune escape, ultimately results in tumor recurrence and turns
It moves.Under one group of participation reconcile stop immune response negativity costimulatory molecules, including PD-1, B7-H1 (PD-L1), B7-H3 and
B7-H4 etc., also known as immune control point, unconventionality expression cause under antitumor immunity of organism power in the different phase of tumor development
Drop promotes growth and metastasis of tumours, and the wherein B7-H1 of negativity B7 families is had found and identified earliest, B7-H1（PD-L1）Belong to
4th member of B7 families, people B7-H1 are the I type transmembrane proteins for encoding 290 amino acid, and IgV samples are included on protein structure
Area, IgC samples area, transmembrane region and a short and conservative cytoplasmic domain.The mRNA of B7-H1 is mainly expressed in placenta, heart（Table in mouse
Up to relatively low）, liver, lung, kidney, the non-lymphoid tissue such as skeletal muscle and a small number of hematopoietic tissue, some prematurity lineage markers it is negative and
The myeloid element and Leukemia Cell Lines of the c-Kit positives also have the expression of B7-H1 mRNA；Thymic tissue has high level
The expression of B7-H1 mRNA, but it is little in the expression quantity of the lymphoid tissues such as lymph node and spleen.On protein level, B7-H1 is wide
General constructive expression organizes, in a variety of organa parenchymatosums on immunocyte and polytype tumour cell, such as inducible expression
In the T, B, monocyte and tumour cell of activation（Such as：Lung cancer, liver cancer, breast cancer, squamous cell carcinoma and oophoroma etc.）.Largely
Research show that B7-H1 can significantly inhibit the biological function of T effector cell with PD-1 interactions in the T cell of activation.
So far, the therapeutic antibodies of FDA approveds targeting PD-1/PD-L1 are for treating metastasis melanin tumor,
Non-small cell lung cancer, clear-cell carcinoma and urothelial carcinoma etc..However, late in metastasis melanin tumor, PD-1 clinical drugs
The validity of application receives very big obstruction.Keytruda and Opdivo resists as the earliest PD-1 for obtaining FDA approval listings
Body drug, mono- line single therapy PD-L1 high of Keytruda expression（PD-L1≥50%）Advanced NSCLC, either PFS or OS
Standard chemotherapeutic group is superior to, the III phases are studied and reach Primary Endpoint and terminate in advance；Mono- line single therapy PD-L1 of Opdivo sun
Property expression（PD-L1≥5%）The III phases of advanced NSCLC study the Primary Endpoint for then failing to reach PFS.Clinical test results are to card
Control point immunization therapy proposes new problem：It needs to detect the expression of PD-1, PD-L1 and definite expression before patient medication
Evaluation criterion.Several clinical testing datas for lung cancer, malignant mela noma and stomach cancer are the results show that with patient PD-L1
The raising of expression, PD-1 drug responses rate, progression free survival phase and median survival interval are significantly improved, and prompt us PD-
L1 is with diagnosis in terms of the antibody medication of immunotherapy of tumors is instructed, improve cancer pathology detection and the accuracy of prognosis evaluation
It is from now on that indispensable key player, the therapeutic monoclonal antibodies of PD-1 and PD-L1, which are play, with the common research and development of diagnostic reagent
The inexorable trend of immune control point targeted therapy.FDA approval 28 kinds with diagnosis (Companion Diagnostics,
CDx) in product, PD-L1 monoclonal antibodies are used for quantitative immune tissue chemical analysis, and testing result will diagnose for oncotherapy and provide ginseng
It examines.PD-L1 expressions and clinical benefit are indicated above there are certain correlation, tumor patient is receiving PD-1 monoclonal antibody medicines
It is necessary to carry out the detection of PD-L1 molecules before treatment.The country there is no high sensitivity, specificity good and can ratify with FDA at present
Two kinds of PD-L1 have the diagnostic reagent of similary detection result with antibody, develop PD-L1 groups detection reagent for judging to target
Whether the therapeutic scheme of PD-1 is applicable in and predicts that patient is most important for the therapeutic effect of certain drug.
The content of the invention
An object of the present invention is to provide a kind of anti-human B7-H1 monoclonal antibodies.
The second object of the present invention is to provide the identification of a kind of anti-human B7-H1 heavy chain of antibody and light chain variable region.
The present invention extracts heavy chain variable region (mVH) and gently from the hybridoma for secreting anti-human B7-H1 monoclonal antibodies
Chain variable region (mVL) retains the heavy and light chain variable region sequence of candidate according to sequencing result, and PCR amplification goes out to be matched with expression vector
Heavy chain, light-chain variable sequence, PCR product is connected with the pretreated linear expression vector of double digestion, connection product turn
Change competent bacteria DH5a.The expression vector cotransfection eukaryotic expression for connecting purposeful monoclonal antibody heavy chain and chain variable region gene is thin
Born of the same parents' strain 293, the supernatant for cultivating harvest contain purposeful antibody, it was demonstrated that obtained heavy chain and light-chain variable sequence is correct；
The third object of the present invention is to provide a kind of anti-human B7-H1 monoclonal antibodies and detects tissue specimen, hair for immunohistochemistry
Current antibody B7-H1 carries out histochemical staining to tonsil, positive expression around lymph follicle；It is further used for exempting from
The Pathologic specimen detection of epidemic disease group, the results show B7-H1 is in the endochylema and nucleus and colon-cancer cell of intestinal cancer patient's interstitial cell
After birth expressed in high.
To achieve these goals, technical scheme is as follows：
As a kind of anti-human B7-H1 monoclonal antibodies of the first aspect of the present invention, including heavy chain and light chain, which is characterized in that institute
It is identical with SEQ ID NO.1 to state the amino acid sequence of heavy chain variable region (mVH), it is specific as follows：
EVQLVESGGDLVKPGGSLKLSCAASGFTFSYYAMSWVRQTPEKRLEWVASISSRGDTYYPDIVKGRFTISRDN
ARNILYLQMRSLRSEDTALYYCARGGGNYEGLDYWGQGTSFTVSS；
The amino acid sequence of the light chain variable region (mVL) is identical with SEQ ID NO.2, specific as follows：
QIVLTQSPAIMSASPGEKVTMTCSVSSSITFMYWFQQKPGSSPRLLIYDTSKLASGVPVRFSGSGSGTSYSLT
ISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIK。
In a preferred embodiment of the invention, heavy chain variable region (mVH) tool is there are three hypervariable region：
GFTFSYYA、ISSRGDT、AARGGGNYEGLDY。
In a preferred embodiment of the invention, light chain variable region (mVL) tool is there are three hypervariable region：SSVGY、
ATS、QQWSSNPPT。
As a kind of anti-human B7-H1 heavy chain of antibody of the second aspect of the present invention and the identification of light-chain variable sequence,
It is characterized in that, comprises the following steps：
（1）The preparation of the hybridoma of B7-H1 monoclonal antibodies；
（1.1）Immune mouse；
（1.2）Cell culture；
（1.3）Fusion and screening；
（2）The preparation of B7-H1 mouse/people's chimeric antibody；
（2.1）Extract the cDNA of hybridoma：RNA is extracted from the hybridoma cell strain, and using RT-PCR technology, will be obtained
The RNA reverse transcriptions obtained are cDNA；
The heavy chain variable region (mVH) and light chain variable region of the hybridoma are cloned using the upstream and downstream primer PCR of particular design
(mVL)；The sequence of the upstream and downstream primer of the particular design is respectively nucleotide SEQ ID NO.3 and nucleotide sequence 4；
（2.2）The heavy chain variable region (mVH) and light chain variable region (mVL) respectively with cloning vector (pJET cloning
Vector) connect, connection product transformed competence colibacillus bacterium DH5a, it, can since pJET carriers are with ammonia benzyl (Amp+) resistant gene
Transformed bacteria solution is coated on the LB solid mediums of Amp resistances, 37 ° are incubated overnight；
（2.3）It treats that coated plate bacterium grows isolated colonies, selects edge clear, well-grown bacterium colony, further sequencing identification；
（2.4）The heavy chain variable region (mVH) of candidate and light chain variable region (mVL) sequence are retained according to sequencing result, PCR expands again
Increase with the matched heavy chain variable region of expression vector (mVH) and light chain variable region (mVL) sequence, PCR product is pre- with double digestion
The linear expression vector's connection first handled, connection product transformed competence colibacillus bacterium DH5a, since expression vector carries kanamycins
(Kana+) transformed bacteria solution can be coated on the LB solid mediums of Kana resistances by resistant gene, and 37 ° are incubated overnight；
（2.5）Sequencing approach reference（2.4）Sequencing result twice is compared, selects the transformed bacteria of correct sequence, it is laggard to expand culture
Row plasmid extraction；
（2.6）The expression vector cotransfection for connecting purposeful monoclonal antibody heavy chain variable region (mVH) and light chain variable region (mVL) gene is true
Nuclear expression cell line 293；
（2.7）The supernatant of harvest contain purposeful antibody, flow cytometer detection expression monoclonal antibody be well combined with M435, positive rate up to 95% with
On, show to have obtained correct heavy chain of antibody and light-chain variable sequence.
In a preferred embodiment of the invention, wherein, step（2.6）In, the eukaryotic expression cell line 293 is outstanding
Floating culture, the passage amplification of SFM4Transfx-293 without L-glutamine (liquid) serum free medium, transfection
When with 293 Expression Medium serum free mediums of Gibco FreeStyle replace；Pass through continuous culture in 7 days
After harvest supernatant, 4000g centrifugation 30min remove the impurity such as cell in supernatant, and with 0.45um filter filtration sterilizations.
As a kind of application of anti-human B7-H1 monoclonal antibodies of the third aspect of the present invention, which is characterized in that described anti-human
B7-H1 monoclonal antibodies are used to prepare the kit of the immunohistochemistry detection for tumor tissues.
A kind of kit as the fifth aspect of the present invention, which is characterized in that the kit includes above-mentioned anti-human B7-
H1 monoclonal antibodies.
Beneficial effects of the present invention：
Anti-human B7-H1 monoclonal antibodies provided by the invention can be used for the immunohistochemistry detection of tumor tissues, improve tumour mark
The accuracy of this immunohistochemistry detection, contributes to the diagnosis of malignant tumour, determines original site and pathological, and it is outstanding to improve tumour
It is the accuracy rate of diagnosis of low differentiation or undifferentiated tumour.
Description of the drawings
Fig. 1 is identifications of the anti-human B7-H1 monoclonal antibodies XGMHc25.1 of flow cytometry to M435 surfaces B7-H1 molecules,
Middle grey peak is negative control.
A is positive control (positive control), and primary antibody is the anti-human B7-H1 monoclonal antibodies of commercialization PE marks.
B is experimental group, and primary antibody is anti-human B7-H1 antibody XGMHc25.1, and secondary antibody is the goat anti-human igg of fluorescein PE marks.
Fig. 2 is that karyotyping, hybridoma caryogram point are carried out to the hybridoma of acquisition using nuclear targeting body technique
It analyses the results show that the chromosome number of hybridoma cell strain is at 80 or more, more than the dyeing of mouse B cell and SP2/0 cells
Body number, it is fused cell to show it.
Fig. 3 detects tonsil, B7-H1 tables around tonsillotome lymph follicle for B7-H1 antibody for immunohistochemistry
It reaches, CST is the antibody of commercialization, and positive expression is had no in the region.
Fig. 4 detects clinical intestinal cancer patient specimen for B7-H1 antibody for immunohistochemistry：B7-H1 is a large amount of in A and B figure cases
Expression is in interstitial cell, based on cytoplasm and nucleus expression；C, D are partial enlarged view.Further illustrate that B7-H1 exists
The after birth of the endochylema of intestinal cancer patient's interstitial cell, nucleus and colon-cancer cell is expressed in high.
The immunohistochemistry that Fig. 5 has carried out B7-H1 molecules to 160 clinical stomach organizations detects, negative for tumor cells coloring；
+, the coloring of 1% ~ 30% tumour cell；++, the coloring of 30% ~ 60% tumour cell；+++,>60% tumour cell colour, score for-and+
Case be classified as low expression group, scoring is ++ and +++ case be classified as high expression group.
Fig. 6 analyzes the CD8 positive cells of negativity costimulatory molecules B7-H1 joint infiltrations, analyzes the prognosis of patient
It has been shown that, B7-H1/CD8 is to have the certain significance for the clinical application guidance of Patients with Gastric Cancer and prognosis evaluation, in the low infiltrations of CD8
In the tissue of B7-H1 high expression, patient's prognosis is poor, and CD8 high is expressed, and patient's prognosis is preferable.
Specific embodiment
Below in conjunction with specific embodiment, make progress explanation to the present invention.It is to be understood that following embodiment is merely to illustrate this hair
It is bright not for limit the scope of the present invention.
In embodiment unless otherwise specified, it is this field conventional laboratory techniques.
Biological material source used is as follows in embodiment：
Embodiment 1 generates the acquisition of the hybridoma of B7-H1 monoclonal antibodies
（One）Immune mouse
Mouse is immunized with fusion protein or transgenic cell, is immunized four times, often minor tick 21 days, is surveyed after the 4th immune 7-10 small
Rathole socket of the eye blood potency, the good then booster immunization of titration.
（Two）Cell culture
1. the 1d before fusion takes 6 ~ 7 week old BALB/c mouse 1, is placed in 2min in 75% ethanol solution.
The sterile taking-up mouse spleens of 2, are placed in the stainless steel mesh of 200 mesh, and grinding obtains individual cells suspension.With
1640 basal mediums wash twice（1400rpm, 5min）It is spare.With 1640 culture mediums of 15%FBS, adjustment cell concentration is 2
×105/ ml is added dropwise in 96 well culture plates, per hole 100 μ L, 37 DEG C, 5%CO2It is cultivated in incubator.
3. being incubated overnight, observed under low-powered microscope within second day.And spread subcloned cells 150ul.
（Three）Fusion and screening
Prepare：
1. equipment：Steep mouse cup；Simple autopsy table；Hybridoma bag；Keep the temperature water-bath cup；Thermometer；96 well culture plates.
2. reagent：75% ethanol solution；37 DEG C of 1640 basal mediums of preheating；37 DEG C of preheating PEG1ml；37 DEG C of preheatings 1640
Basal medium 14ml（PEG terminate liquids）；37 DEG C of 1640 Selective agar mediums of preheating（Containing 15%FBS, and the amount for adding in HAT is calculated,
So that in final 96 well culture plate culture medium HAT final concentration of 1%）.
Step：
It 1. by immune mouse, is rinsed with flowing water, is placed in 2min in 75% ethanol solution.
2. sterile taking-up mouse spleen, is placed in the stainless steel mesh of 200 mesh, grinding obtains individual cells suspension.With pre-
Hot 1640 basal mediums wash twice（1400rpm, 5min）It is spare.
3. collecting well-grown, the SP2/0 cells in exponential phase, two are washed with 1640 basal mediums of preheating
Time（1400rpm, 5min）It is spare.
4. SP2/0 cells or Ag8 cells are mixed in spleen cell in 50ml transparent plastic centrifuge tubes, general spleen is thin
Born of the same parents:Myeloma cell's ratio is 5:1,1640 basal mediums of preheating wash one time（1400rpm, 5min）, abandon most supernatant（To keep away
Exempt to generate unnecessary dilution to PEG）And with centre of the palm dematron bottom（Or finger gently attack tube bottom）, make two kinds of abundant mixings of cell
Into suspension cell shape.
It is preheated 5. centrifuge tube is placed in 37 DEG C of heat preservation water-bath cups, draws 50% PEG solution of the 1ml through 37 DEG C of pre-temperatures,
It is at the uniform velocity added in 1min, and side edged gently shakes centrifuge tube, gently shakes 60s in 37 DEG C of water-baths after adding.（It drips within 3 seconds）
6. 1640 basal mediums that 37 DEG C of pre-temperatures of 14ml are softly added in along tube wall terminate（1min adds 1ml, 3min to add
3ml is finally slowly added to 10 ml）.（At the uniform velocity it is added dropwise）
7. it is centrifuged after 37 DEG C of static 5min（800rpm, 5min）, supernatant discarding（Centrifuge tube is tilted, sucks supernatant）.
8. sedimentation cell gently is resuspended (can not blow and beat) with 1640 Selective agar mediums of 37 DEG C of preheatings, it is added in advance
After in ready preheating culture medium, it is added dropwise after mixing in above-mentioned 96 well culture plates containing trophocyte, 100 μ l/ holes are put
It is cultivated in 37 DEG C, 5%CO2 incubators, the later half amounts of 3-4d change liquid, use HT medium cultures after 10d instead, and conversion is containing 10% after 2 weeks
1640 medium cultures of FBS.
9. clonal growth situation in 96 orifice plates is observed during daily, is generally covered with 1/10 area of bottom hole in hybridoma
When, you can start to detect specific antibody, filter out required hybridoma cell line.For there is the thin of specific secretion antibody
Born of the same parents should clone in time to be cultivated and freezes.Usually need 3-5 times subclone could obtain stabilization genotype and stably excreting phenotype it is thin
Born of the same parents, and need to be subcloned again after cultivating a period of time.
The sequencing of 2 B7-H1 antibody of embodiment weight light chain variable region
1. extract the cDNA of hybridoma：RNA is extracted from the hybridoma cell strain, and using RT-PCR technology, will be obtained
RNA reverse transcriptions be cDNA；The hybridoma heavy chain variable region (mVH) is cloned using the upstream and downstream primer PCR of particular design
With light chain variable region (mVL)；
2. mVH and mVL are connected respectively with cloning vector (pJET cloning vector), connection product transformed competence colibacillus bacterium
Transformed bacteria solution since pJET carriers carry ammonia benzyl (Amp+) resistant gene, can be coated in the LB solid mediums of Amp resistances by DH5a
On, 37 ° are incubated overnight；
3. treating that coated plate bacterium grows isolated colonies, edge clear, well-grown bacterium colony, further sequencing identification are selected；
4. retaining the heavy and light chain variable region sequence of candidate according to sequencing result, PCR amplification goes out matched heavy with expression vector again
PCR product is connected by chain, light-chain variable sequence with the pretreated linear expression vector of double digestion, connection product conversion sense
By state bacterium DH5a, since expression vector is with kanamycins (Kana+) resistant gene, transformed bacteria solution can be coated in Kana resistances
LB solid mediums on, 37 ° are incubated overnight；
5. sequencing approach compares sequencing result twice with reference to 4., the transformed bacteria of correct sequence is selected, matter is carried out after expanding culture
Grain extracting；
6. the expression vector cotransfection eukaryotic expression cell line 293 of the purposeful monoclonal antibody heavy chain of connection and chain variable region gene.
293 cells are cultivated to suspend, and SFM4Transfx-293 without L-glutamine (liquid) serum free medium passes
Generation amplification, is replaced with 293 Expression Medium serum free mediums of Gibco FreeStyle during transfection.Pass through
Supernatant is harvested after continuous culture in 7 days, 4000g centrifugation 30min remove the impurity such as cell in supernatant, and filtered with 0.45um filters
Degerming；
7. the supernatant of harvest is well combined containing purposeful antibody, flow cytometer detection expression monoclonal antibody with breast cancer cell M435.
The B7-H1 heavy chain of antibody variable region obtained by the above method：EVQLVESGGDLVKPGGSLKLSCAASGFTFSY
YAMSWVRQTPEKRLEWVASISSRGDTYYPDIVKGRFTISRDNARNILYLQMRSLRSEDTALYYCARGGGNYEGLDYW
GQGTSFTVSS
Three hypervariable regions：GFTFSYYA、ISSRGDT、AARGGGNYEGLDY.
Antibody light chain variable region：
QIVLTQSPAIMSASPGEKVTMTCSVSSSITFMYWFQQKPGSSPRLLIYDTSKLASGVPVRFSGSGSGTSYSLT
ISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIK
Three hypervariable regions：SSVGY、ATS、QQWSSNPPT.
3 hybridoma chromosome karyotype analysis of embodiment
Well-grown cell is taken, colchicine is added in, makes its final concentration of 0.04 ~ 0.08 μ l/ml.Cell is collected after culture 2h
（1000rpm, 10min）In centrifuge tube, after the 0.075mol/L KCl solution of 37 DEG C of pre-temperatures of 0.5ml is added dropwise, immediately again
5 ~ 10ml is added, is gently blown and beaten with suction pipe uniformly, 37 DEG C of incubation 20min.The fixer of 1ml Fresh is added in pipe（3 parts
Methanol adds 1 part of glacial acetic acid, prepared before use）, 1000rpm, centrifugation 10min abandon supernatant.8 ~ 10ml fixers are added, use suction pipe
Piping and druming is uniform, fixes 15 ~ 20min, centrifuges, abandons supernatant.Add 5ml fixers, fixed 30min.Supernatant is abandoned in centrifugation, is added
1.5ml fixers, piping and druming are uniform.The glass slide of -10 DEG C of frosts is taken, 1 ~ 2 drop cell suspension is added dropwise, with fresh Giemsa solution（1
Part Giemsa stostes add 9 parts of 0.075mol/L, pH6.8 phosphate buffers）10 ~ 20min is dyed, flowing water dries after rinsing.Two
Toluene is transparent three times, resinene mounting.Selective staining body is well dispersed under the microscope, is not overlapped, the sample that nothing is scattered, in
It is observed and recorded under oil mirror and carries out photomicrography.
4 immunohistochemistry technology of embodiment
1. paraffin section
Flesh tissue is taken, is trimmed to 1cm × 1cm × 0.3cm size tissue blocks, is fixed in neutral formalin liquid for 24 hours, gradient wine
Smart solution dehydrates（70% alcohol 15min, 80% alcohol 15min, 90% alcohol 1h, 95% alcohol 2h, absolute ethyl alcohol 2h）, dimethylbenzene is saturating
It is bright（Altogether twice, each each 30min）, waxdip（Altogether twice, first time 1h, second of 2h）, 5 μm of thin slices are cut after paraffin embedding, are pasted
Invest the slide of coating poly-D-lysine.
2. immunohistochemical staining
0.03%H2O2- methanol is incubated 30 minutes, and 10%BSA is added dropwise, and is incubated at room temperature 30 minutes, is not washed, and primary antibody is added dropwise（It is anti-human
CD105 antibody dilutions are 1：100）, replacing primary antibody using PBS, 37 DEG C of incubation 2h, PBS flushings are added dropwise general as negative control
Type secondary antibody is i.e. with liquid, and 37 DEG C are incubated 30min, and PBS is rinsed three times, and DAB-H2O2 colour developings are redyed, 1% hydrochloric acid-alcohol through haematoxylin
Break up, be dehydrated in graded ethanol（75% ethyl alcohol 3min, 95% ethyl alcohol I 3min, 95% ethyl alcohol II 3min, 100% absolute ethyl alcohol
I 3min, 100% absolute ethyl alcohol II 3min）Afterwards, hair-dryer drying absolute ethyl alcohol, resinene mounting, micro- Microscopic observation is simultaneously
It takes pictures.
SEQUENCE LISTING
<110>Suzhou Xu Guang science-star Bioisystech Co., Ltd
<120>The preparation method of anti-human B7-H1 monoclonal antibodies and its application in immunohistochemistry detection
<130> 20161023
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 118
<212> PRT
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> High variable region
<222> (26)..(33)
<220>
<221> High variable region
<222> (51)..(57)
<220>
<221> High variable region
<222> (96)..(107)
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu
1 5 10 15 20
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr Ala Met Ser Trp Val Arg Gln Thr
25 30 35 40
Pro Glu Lys Arg Leu Glu Trp Val Ala Ser Ile Ser Ser Arg Gly Asp Thr Tyr Tyr Pro
45 50 55 60
Asp Ile Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu
65 70 75 80
Gln Met Arg Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Gly Gly Gly
85 90 95 100
Asn Tyr Glu Gly Leu Asp Tyr Trp Gly Gln Gly Thr Phe Ser Thr Val Ser Ser
105 110 115
<210> 2
<211> 106
<212> PRT
<213> Artificial sequence
<220>
<223> Artificial sequence
<220>
<221> High variable region
<222> (27)..(31)
<220>
<221> High variable region
<222> (49)..(51)
<220>
<221> High variable region
<222> (88)..(96)
<400> 2
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr
1 5 10 15 20
Met Thr Cys Ser Val Ser Ser Ser Ile Thr Phe Met Tyr Trp Phe Gln Gln Lys Pro Gly
25 30 35 40
Ser Ser Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Val Arg
45 50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Gly Tyr Pro Tyr Thr Phe Gly Gly Gly
85 90 95 100
Thr Lys Leu Glu Ile Lys
105
Claims (7)
1. a kind of anti-human B7-H1 monoclonal antibodies, including heavy chain and light chain, which is characterized in that the heavy chain variable region (mVH)
Amino acid sequence is identical with SEQ ID NO.1, specific as follows：
EVQLVESGGDLVKPGGSLKLSCAASGFTFSYYAMSWVRQTPEKRLEWVASISSRGDTYYPDIVKGRFTISRDN
ARNILYLQMRSLRSEDTALYYCARGGGNYEGLDYWGQGTSFTVSS；
The amino acid sequence of the light chain variable region (mVL) is identical with SEQ ID NO.2, specific as follows：
QIVLTQSPAIMSASPGEKVTMTCSVSSSITFMYWFQQKPGSSPRLLIYDTSKLASGVPVRFSGSGSGTSYSLT
ISRMEAEDAATYYCQQWSGYPYTFGGGTKLEIK。
A kind of 2. anti-human B7-H1 monoclonal antibodies as described in claim 1, which is characterized in that the heavy chain variable region
(mVH) tool is there are three hypervariable region：GFTFSYYA、ISSRGDT、AARGGGNYEGLDY.
A kind of 3. anti-human B7-H1 monoclonal antibodies as described in claim 1, which is characterized in that the light chain variable region (mVL)
There are three tools, and hypervariable region is：SSVGY、ATS、QQWSSNPPT.
4. a kind of preparation method of anti-human B7-H1 monoclonal antibodies described in any one of claims 1 to 3 claim, special
Sign is, comprises the following steps：
（1）The preparation of the hybridoma of B7-H1 monoclonal antibodies；
（1.1）Immune mouse；
（1.2）Cell culture；
（1.3）Fusion and screening；
（2）The preparation of B7-H1 mouse/people's chimeric antibody；
（2.1）Extract the cDNA of hybridoma：RNA is extracted from the hybridoma cell strain, and using RT-PCR technology, will be obtained
The RNA reverse transcriptions obtained are cDNA；
The heavy chain variable region (mVH) and light chain variable region of the hybridoma are cloned using the upstream and downstream primer PCR of particular design
(mVL)；The sequence of the upstream and downstream primer of the particular design is respectively nucleotide SEQ ID NO.3 and nucleotide sequence 4；
（2.2）The heavy chain variable region (mVH) and light chain variable region (mVL) respectively with cloning vector (pJET cloning
Vector) connect, connection product transformed competence colibacillus bacterium DH5a, it, can since pJET carriers are with ammonia benzyl (Amp+) resistant gene
Transformed bacteria solution is coated on the LB solid mediums of Amp resistances, 37 ° are incubated overnight；
（2.3）It treats that coated plate bacterium grows isolated colonies, selects edge clear, well-grown bacterium colony, further sequencing identification；
（2.4）The heavy chain variable region (mVH) of candidate and light chain variable region (mVL) sequence are retained according to sequencing result, PCR expands again
Increase with the matched heavy chain variable region of expression vector (mVH) and light chain variable region (mVL) sequence, PCR product is pre- with double digestion
The linear expression vector's connection first handled, connection product transformed competence colibacillus bacterium DH5a, since expression vector carries kanamycins
(Kana+) transformed bacteria solution can be coated on the LB solid mediums of Kana resistances by resistant gene, and 37 ° are incubated overnight；
（2.5）Sequencing approach reference（2.4）Sequencing result twice is compared, selects the transformed bacteria of correct sequence, it is laggard to expand culture
Row plasmid extraction；
（2.6）The expression vector cotransfection for connecting purposeful monoclonal antibody heavy chain variable region (mVH) and light chain variable region (mVL) gene is true
Nuclear expression cell line 293；
（2.7）The supernatant of harvest contain purposeful antibody, flow cytometer detection expression monoclonal antibody be well combined with M435, positive rate up to 95% with
On, it was demonstrated that above-mentioned heavy chain and light-chain variable sequence are correct.
5. the preparation method described in claim 4, which is characterized in that step（2.6）In, the eukaryotic expression cell line 293 is
Suspend culture, and the passage amplification of SFM4Transfx-293 without L-glutamine (liquid) serum free medium turns
It is replaced during dye with 293 Expression Medium serum free mediums of Gibco FreeStyle；Pass through continuous training in 7 days
Supernatant is harvested after supporting, 4000g centrifugation 30min remove the impurity such as cell in supernatant, and with 0.45um filter filtration sterilizations.
6. a kind of application of anti-human B7-H1 monoclonal antibodies described in any one of claims 1 to 3 claim, feature exist
In the anti-human B7-H1 monoclonal antibodies are used to prepare the kit of the immunohistochemistry detection for tumor tissues.
A kind of 7. kit, which is characterized in that the anti-human B7- described in any one of described kit claims 1 to 3 claim
H1 monoclonal antibodies.
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| CN102250911A (en) * | 2011-05-31 | 2011-11-23 | 苏州大学 | Soluble B7-H1 quantitative detection kit |
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| WO2019237442A1 (en) * | 2018-06-15 | 2019-12-19 | 苏州旭光科星生物技术有限公司 | Anti-human b7-h4 monoclonal antibody, identification method, use and kit |
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