CN106854244A - A kind of nano antibody and its clinical practice for HER3 - Google Patents
A kind of nano antibody and its clinical practice for HER3 Download PDFInfo
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Abstract
The invention discloses a kind of VHH chains of the nano antibody of HER3, including framework region FR and complementary determining region CDR, disclose the amino acid sequence and complementary determining region cdr amino acid sequence of the FR that framework region FR is selected from the group, the invention also discloses a kind of HER3 nano antibodies, also disclose a kind of DNA molecular, it encodes the VHH chains or HER3 nano antibodies of the present invention of the nano antibody of HER3 of the present invention, also disclose a kind of host cell, it can express the nano antibody of HER3, also disclose the HER3 nano antibodies for detecting the purposes of HER3.By the nano antibody gene order announced of the present invention and host cell, the nano antibody can in Escherichia coli high efficient expression, be applied to the research and development of HER3 detection reagents and bio-pharmaceuticals.
Description
Technical field
The invention belongs to biomedical or biological pharmacy technical field, it is related to be directed to the nano antibody and its coding of HER3
Sequence.
Background technology
Belgium scientist Hamers.R has found in camel body within 1993, compared with conventional antibodies, in camel blood
Antibody, there is half there is no light chain, only include a weight chain variable district（VHH）It is current with two CH2 and the CH3 areas of routine
It is known can combining target antigen least unit.VHH crystal diameters are 2.5nm, and 4nm long, molecular weight only has 15KD, therefore
It is referred to as nano antibody（Nanobody, Nb）.Compared with conventional antibody, the VHH single domain antibodies based on camel heavy chain antibody have
Many advantages：1）Molecular weight is small, can penetrate blood-brain barrier, is easier to act on focal zone；2）Can in protokaryon or eukaryotic system
Height expression；3）High specificity, affinity are high；4）High-fire resistance and chemical stability；5）Immunogenicity to people is weak.It is swollen
Diagnosis and the treatment aspect of knurl, infectious diseases, enteritis, amyloidosis diseases, thrombus and atherosclerotic lesion have
Good application effect, while cost can be significantly reduced.
In China, the health burden of cancer increases year by year, and cancer is diagnosed as per year over 1600000 people, and 1,200,000 people are because of cancer
And it is dead.As other most countries, breast cancer also becomes the most common cancer of Chinese women, and it is each that the incidence of disease accounts for whole body
Plant the 7 ~ 10% of malignant tumour；Annual Chinese Breast Cancer newly sends out quantity and The dead quantity and accounts for global 12.2% and 9.6% respectively,
It is HER3 overexpression that 1/4 is there are about in patient with breast cancer.Current conventional detection is to carry out qualitative, semidefinite to the biopsy of patient
Amount method SABC（IHC）, but this method is time-consuming more long and cumbersome.Therefore in order in time to the spy of breast cancer HER3 expression
Opposite sex detection overexpression patient carries out individuation, targeting and effectively treats, and prepares breast cancer overexpression epidermal growth factor receptor
Body 3（HER3）Nano antibody come carry out disease detection and treatment have broad prospects.
At present, also it is not directed to breast cancer overexpression EGF-R ELISA 3（HER3）It is the specific nano of target
The research report of antibody.
The content of the invention
Goal of the invention：The technical problems to be solved by the invention are to provide a kind of nano antibody for HER3, while carrying
The application of detection is being prepared for the coded sequence of the nano antibody and the nano antibody.
Technical scheme：The technical scheme is that：
The first aspect of the present invention, there is provided one kind is directed to HER3 heavy chain antibody VHH, including framework region FR and complementary determining region
CDR, the amino acid sequence of the FR that the framework region FR is selected from the group：SEQ ID NO：FR1 shown in 1, SEQ ID NO：Shown in 2
FR2, SEQ ID NO：FR3 shown in 3, SEQ ID NO：FR4 shown in 4.
The amino acid sequence of the CDR that the complementary determining region CDR is selected from the group：SEQ ID NO：CDR1 shown in 5, SEQ
ID NO：CDR2 shown in 6, SEQ ID NO：CDR3 shown in 7.
Preferably, the VHH chains of described HER3 nano antibodies, it has SEQ ID NO：Amino acid sequence shown in 8.
Second aspect present invention, one kind is directed to HER3 heavy chain antibody VHH, and this antibody specificity recognizes HER3 antigens, including
One has SEQ ID NO：The VHH chains of amino acid sequence shown in 8.
A kind of third aspect present invention, there is provided DNA molecular, it encodes the protein being selected from the group：It is of the present invention
HER3 heavy chain antibodies VHH.
Preferably, described DNA molecular, it is characterised in that it has the DNA sequence dna being selected from the group：SEQ ID NO：9.
Beneficial effect：Compared with prior art, advantages of the present invention is as follows：The HER3 that the present invention will be extracted in blood is immunized
Xinjiang one-humped camel, the nano antibody gene pool for being directed to HER3 is established followed by the camel PBLC, experiment
Middle that HER3 is coupled on ELISA Plate, antigen in this format utilizes display technique of bacteriophage to screen the nano antibody base of immunity
Yin Ku（Camel heavy chain antibody phage display gene pool）, so as to obtain for the specific nano antibody genes of HER3, will
This gene is gone in Escherichia coli, can be in the strain of the nano antibody of E. coli so as to establish.
Brief description of the drawings
Fig. 1 is the gene electrophoretogram of HER3 nano antibodies；Wherein swimming lane 1 is DNA molecular standard, and swimming lane 2 is PCR amplification weights
Chain antibody guides the fragment between peptide and antibody CH2, and PCR primer band is about 700bp.
Fig. 2 is the DNA agarose gel electrophoresis figures of HER3 nano antibodies, and from left to right the DNA bands of gel pore are respectively：
First is the molecular labeling of 1000bp, and remaining duct is that PCR expands antibody heavy chain variable region fragment products, PCR primer band
About 500bp；
Fig. 3 is the insertion rate testing result in the single domain antibody library for building, and from left to right the DNA bands of gel pore are respectively：
First is DNA molecular marker, and remaining duct is the PCR primer for detecting Insert Fragment, and after testing, the insertion rate in the library reaches
More than 90%.
Fig. 4 is with the enzyme-linked immunoassay method of bacteriophage（ELISA）Screen the ideograph of specific single positive colony；Wherein
1 is that by HER3 albumen couplings on ELISA Plate, 2 is nano antibody, and 3 is the anti-HA antibody of mouse, and 4 is goat-anti-mouse alkaline phosphatase
The antibody of mark, 5 is alkaline phosphatase nitrite ion.
Fig. 5 is the HER3 nano antibodies of expression, through the electrophoresis of the SDS-PAGE after nickel post resin gel affinitive layer purification
Figure；Wherein swimming lane 1 is protein molecular standard, and remaining swimming lane is that the nanometer that 250 mMs of imidazole elutions are eluted resists
Body, as a result shows HER3 nano antibodies by the purge process, and its purity can reach more than 95%.
Fig. 6 is the result figure of HER3 nano antibodies detection specificity analysis.
Specific embodiment
The present invention using the soluble protein of HER3 as one Xinjiang one-humped camel of antigen immune, it is immunized by 5 times first
The one-humped camel PBLC is extracted afterwards and constructs the special single domain heavy chain antibody libraries of HER3.HER3 is coupled at
On NUNC ELISA Plates, the correct space structure of display protein matter so that the epitope of HER3 is exposed, in this format
Antigen using display technique of bacteriophage screen HER3 immunitys nano antibody gene pool（Camel heavy chain antibody phage display
Gene pool）, and obtain can E. coli nano antibody strain.
With reference to specific embodiment, the present invention is expanded on further.
Embodiment 1
It is directed to the structure in the nano antibody library of HER3：
（1）Synthetic antigen HER3 first, the concentration for HER3 used to be immunized is 500 μ g/mL, and being immunized every time will
0.5mgHER3 mixes in equal volume with Freund's adjuvant, an Xinjiang one-humped camel (Jurong Sheng Long livestock culturings factory) is immunized, on every Mondays
It is secondary, it is immunized 5 times altogether, except first time uses complete Freund's adjuvant（Purchased from sigma）, it is remaining not helped entirely using not formula all several times
Agent（Purchased from sigma）, the nano antibody of immunologic process moderate stimulation B cell expression antigentic specificity.（2）After 5 immune end, carry
Take camel PBLC 100mL and extract the RNA extracts kits that total serum IgE is provided with reference to QIAGEN companies.（3）According to
Super-Script III FIRST STRANDSUPERMIX kit specifications, the RNA reverse transcriptions that will be extracted are into cDNA.With
The variable region fragment of nested PCR amplification heavy chain antibody：
First round PCR：
Sense primer GTCCTGGCTGCTCTTCTACAAGGC
Downstream:GGTACGTGCTGTTGAACTGTTCC
Fragment between amplification heavy chain antibody guiding peptide and antibody CH2,54 DEG C of annealing, 25 circulations；Result such as Fig. 1 shows the piece
The size of section is about 700bp, i.e., DNA bands from left to right are respectively：First is the molecule Marker of DNA, and the second single domain resists
Body gene electrophoresis band is about 700bp.
Second wheel PCR：
Template is made with first round PCR primer,
Sense primer：GATGTGCAGCTGCAGGAGTCTGGRGGAGG
Anti-sense primer：GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT
Fragment between amplification heavy chain antibody FR1 areas and long and short hinge area（Long segment and short-movie section）, 60 DEG C of annealing, 17 are followed
Ring, reclaims purpose fragment, as a result as Fig. 2 shows that the size of the fragment is about 500bp, i.e., DNA bands difference from left to right
It is：First is DNA molecular Marker, and the second nano antibody gene electrophoresis band is about 500bp.（4）Use restricted restriction endonuclease
（Purchased from NEB）The μ g pComb3 Vector for Phage Display of PstI and NotI digestions 20（Biovector is supplied）And 10 μ g VHH be used in combination
T4 DNA ligases（Purchased from TaKaRa companies）Two fragments of connection.（5）Connection product electricity conversion to electricity is turned into competent cell
TG1（Beijing Divine Land red autumnal leaves Science and Technology Ltd.）In, build the nano antibody phage display library of HER3 and determine storage capacity, storehouse
The size of appearance is 3.1 × 108.At the same time, the second wheel PCR primer, Tm55 DEG C are used by bacterium colony PCR detection primers.Library
After the completion of structure, to detect the insertion rate in library, randomly select single clone and be bacterium colony PCR.Result such as Fig. 3 display insertions rate is
Reach more than 90%.
Embodiment 2
For the nano antibody screening process of HER3：
（1）The HER3 of 100 μ g/mL being dissolved in PBS is coated on NUNC ELISA Plates, and 4 DEG C stand overnight, while setting up negative
Control.（2）200 μ L1% milk are separately added into second day two hole, room temperature is closed 2 hours.（3）After 2 hours, 100 μ L are added
Bacteriophage（8×1011The immune camel nano antibody phage display gene pools of tfu）, act on 1 hour at room temperature.(4）Use PBST
（Contain 0.05% polysorbas20 in PBS）Wash 5 times, to wash uncombined bacteriophage off.(5）Use triethylamine（100mM）Will be with HER3
Under the bacteriophage dissociation of specific binding, and the e. coli tg1 in logarithmic phase growth is infected, produce and purified phage is used
In the screening of next round, identical screening process repeats 2 and takes turns.Result such as Fig. 4 shows：It is positive during constantly screening
Clone will be constantly enriched with, and take the mesh of HER3 specific antibodies in antibody library using display technique of bacteriophage sieve so as to reach
's.
Embodiment 3
With the enzyme-linked immunoassay method of bacteriophage（ELISA）Screen specific single positive colony：
The principle modes figure of the experiment is as shown in figure 4, specifically detect as follows：
（1）From the Tissue Culture Dish containing bacteriophage after 3-4 wheel screenings, select 96 single bacterium colonies and be inoculated in and contain 100 μ
The ampicillin of g/mL TB culture mediums (in 1L TB culture mediums contain 2.3g potassium dihydrogen phosphates, 12.52g dipotassium hydrogen phosphates,
12g peptones, 24g yeast extracts, 4mL glycerine) in, after growing to logarithmic phase, plus final concentration 1mmol IPTG, 28 DEG C training
Support overnight.（2）Obtained using osmosis and slightly carry antibody, and antibody is transferred in the elisa plate through antigen coat, at room temperature
Place 1 hour.（3）Uncombined antibody is washed away with PBST, primary antibody mouse anti-HA tag antibody are added（Anti- mouse resists
HA antibody, purchased from Beijing CoWin Bioscience Co., Ltd.）, place 1 hour at room temperature.（4）Washed away with PBST and do not tied
The antibody of conjunction, adds secondary antibody anti-mouse alkaline phosphatase conjugate（Goat-anti-mouse alkaline phosphoric acid
Enzymic-labelled antibody, purchased from Amy victory Science and Technology Ltd.）, place 1 hour at room temperature.（5）Uncombined resisting is washed away with PBST
Body, adds alkaline phosphatase nitrite ion, on ELISA instrument, in 405nm wavelength, reads absorption value.（6）When sample well OD values are big
When more than 3 times of control wells OD values, positive colony hole is judged to.（7）The bacterium in positive colony hole is turned to shake to contain 100 μ g/mL's
To extract plasmid and to be sequenced in LB liquid.
The gene order of each clone strain is analyzed according to sequence alignment program Vector NTI, CDR1, CDR2, CDR3 sequence phase
Same strain is considered as same clone strain, and the different strain of its sequence is considered as different clone strains, finally gives amino acid sequence SEQ ID
NO：Nano antibody shown in 8.
Embodiment 4
Nano antibody is in Host Strains expression in escherichia coli, purifying：
（1）Above sequencing analysis are obtained into two kinds of nano antibodies to be subcloned into the carrier PET32a of expressivity, and will sequencing
Correct recombinant plasmid transformed is identified in expression type Host Strains DE3, it is coated on the LB containing 100 μ g/mL ampicillins
On the plate of solid medium, 37 DEG C overnight.（2）Select single bacterium colony and be seeded in the LB nutrient solutions that 15mL contains ampicillin
In, 37 DEG C of shaking table cultures are overnight.（3）It is inoculated with overnight strain to the 330mLLB culture mediums of 1mL, 37 DEG C of shaking table cultures, culture is arrived
When OD values reach 0.6-1, IPTG is added, 28 DEG C of shaking table cultures are overnight.（4）Second day, bacterium was received in centrifugation.（5）By bacterial cell disruption with
Obtain antibody crude extract.（6）Through nickel post ion affinity chromatography antibody purification albumen, to obtain the antibody of high-purity, using imidazoles
Linear gradient elution method, low concentration imidazole elution（50mmol）For washing away miscellaneous band, high concentration imidazole elution（250mmol,
500mmol）The albumen that purity is up to more than 90% can finally be prepared.Band shown in Fig. 5 from left to right is respectively：First is standard
Protein molecular, the protein sample of the elution of the 2nd the 3rd 250mmol imidazoles；Result shows that nano antibody is pure by this
After change, its purity can reach more than 95%.
Embodiment 5
Biotinylation nano antibody and its purification process：
（1）Will be directed to HER3 nano antibody genetic fragment be subcloned to pBAD carriers, the plasmid pBAD for then building with
Plasmid BirA corotation is coated on the LB cultures containing ampicillin, chloramphenicol and glucose in Escherichia coli WK6
On flat board, 37 DEG C of overnight incubations；（2）Select single bacterium colony and be seeded in the LB nutrient solutions that 5mL contains ampicillin and chloramphenicol
In, 37 DEG C of shaking table cultures are overnight；（3）The overnight strain for being inoculated with 1mL contains the TB nutrient solutions of ampicillin and chloramphenicol to 330mL
In, 37 DEG C of shaking table cultures when culture to OD values reaches 0.4-0.5, add the Bio of 330 μ l50mM（D-biotion）It is molten
Liquid, 37 DEG C are shaken 1h slowly；（4）Final concentration of 1mMIPTG is added, 28 DEG C of shaking table cultures are overnight；（4）Centrifugation, receives bacterium；（5）Using oozing
Saturating method, obtains antibody crude extract；（6）The nano antibody of couple biotin is purified using Streptavidin MagneSphere.
Embodiment 6
The detection specificity analysis of HER3 nano antibodies：
（1）HER3 is coated on ELISA Plate, while doing blank well control, two holes is respectively coated with, by HER3 protein nano antibody
And control antibodies prealbumin nano antibody is transferred in the elisa plate through antigen coat respectively, place 1 hour at room temperature.
（2）Uncombined antibody is washed away with PBST, primary antibody mouse anti-HA tag antibody are added（The anti-anti- HA antibody of mouse, purchase
From Beijing CoWin Bioscience Co., Ltd.）, place 1 hour at room temperature.（3）Uncombined antibody is washed away with PBST,
Add secondary antibody anti-mouse alkaline phosphatase conjugate（Goat-anti-mouse alkaline phosphatase enzyme mark resists
Body, purchased from Amy victory Science and Technology Ltd.）, place 1 hour at room temperature.（4）Uncombined antibody is washed away with PBST, alkali is added
Acid phosphatase nitrite ion, on ELISA instrument, in 405nm wavelength, reads absorption value.Result such as Fig. 6 shows HER3 nano antibody energy
Specific identification HER3 albumen.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
The present invention provides a HER3 nano antibody, and each nano antibody provides skeleton area and complementary determining region, wherein described
The nucleotides and amino acid sequence of skeleton area FR1, FR2, FR3, FR4 and complementary determining region CDR1, CDR2, CDR3 are respectively：
HER3 nano antibodies
Skeleton area and the nucleotide sequence of complementary determining region：
FR1:CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCCCGGTGCAGTCTGGAGGGTCTCTGAGACTCTCCTGTGCA
GCCTCT
FR2: TGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGGGTCGCAACT
FR3:GTCTACGCAGACTCCGTGAAGGGCCGATTTACCATCTCCCGAGACGACGCCAAGACGAATCTGTTTCTG
CAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTACTGT
FR4: TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATAC
CDR1: GGAAACATCTACAGTAGGCACTCCATGGGC
CDR2: ATTAATCGTGATGGCGAGATA
CDR3: GCCGCTAAACCAGGCTGGATGGCGACCCTGGCGTGGAGCGACTGGTTTTAC
Skeleton area and the amino acid sequence of complementary determining region：
FR1: QVQLQESGGGPVQSGGSLRLSCAAS
FR2: WFRQAPGKEREGVAT
FR3: VYADSVKGRFTISRDDAKTNLFLQMNSLKPEDSAMYYC
FR4: WGQGTQVTVSSAAG
CDR1: GNIYSRHSMG
CDR2: INRDGEI
CDR3: AAKPGWMATLAWSDWFY
Overall nucleotide sequence：
CAGGTGCAGCTGCAGGAGTCTGGGGGAGGCCCGGTGCAGTCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCT
CTGGAAACATCTACAGTAGGCACTCCATGGGCTGGTTCCGCCAGGCTCCAGGAAAGGAGCGCGAGGGGGTCGCAACT
ATTAATCGTGATGGCGAGATAGTCTACGCAGACTCCGTGAAGGGCCGATTTACCATCTCCCGAGACGACGCCAAGAC
GAATCTGTTTCTGCAAATGAACAGCCTGAAACCTGAGGACAGTGCCATGTACTACTGTGCCGCTAAACCAGGCTGGA
TGGCGACCCTGGCGTGGAGCGACTGGTTTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGCGGCCGCATAC
Overall amino acid sequence：QVQLQESGGGPVQSGGSLRLSCAASGNIYSRHSMGWFRQAPGKEREGVATINRDGEIV
YADSVKGRFTISRDDAKTNLFLQMNSLKPEDSAMYYCAAKPGWMATLAWSDWFYWGQGTQVTVSSAAG
Claims (5)
- The VHH chains of 1.HER3 nano antibodies, including framework region FR and complementary determining region CDR, what the framework region FR was selected from the group The amino acid sequence of FR：SEQ ID NO：FR1 shown in 1, SEQ ID NO：FR2 shown in 2, SEQ ID NO：FR3 shown in 3, SEQ ID NO： FR4 shown in 4；The amino acid sequence of the CDR that the complementary determining region CDR is selected from the group：SEQ ID NO：CDR1 shown in 5, SEQ ID NO：CDR2 shown in 6, SEQ ID NO：CDR3 shown in 7.
- 2. HER3 heavy chain antibodies VHH according to claim 1, it is characterised in that it has SEQ ID NO：Ammonia shown in 8 Base acid sequence.
- 3. the heavy chain antibody VHH of a kind of HER3, it is characterised in that it is directed to the nano antibody of HER3 surface antigens, including has SEQ ID NO：The VHH chains of amino acid sequence shown in 8.
- 4. a kind of DNA molecular, it is characterised in that it encodes the protein being selected from the group：HER3's described in claim 1 or 2 Heavy chain antibody VHH.
- 5. DNA molecular according to claim 4, it is characterised in that it has the DNA sequence dna being selected from the group：SEQ ID NO：9.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110396127A (en) * | 2019-03-21 | 2019-11-01 | 南京东极医药科技有限公司 | The preparation of anti-CD20 nano antibody |
| CN112239504A (en) * | 2020-09-10 | 2021-01-19 | 哈尔滨博易诚生物科技有限公司 | Nano antibody aiming at PD-L1 and application thereof |
| CN112250765A (en) * | 2020-09-10 | 2021-01-22 | 哈尔滨博易诚生物科技有限公司 | Nano antibody aiming at HER2 and application thereof |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110396127A (en) * | 2019-03-21 | 2019-11-01 | 南京东极医药科技有限公司 | The preparation of anti-CD20 nano antibody |
| CN112239504A (en) * | 2020-09-10 | 2021-01-19 | 哈尔滨博易诚生物科技有限公司 | Nano antibody aiming at PD-L1 and application thereof |
| CN112250765A (en) * | 2020-09-10 | 2021-01-22 | 哈尔滨博易诚生物科技有限公司 | Nano antibody aiming at HER2 and application thereof |
| CN114409787A (en) * | 2022-02-18 | 2022-04-29 | 南京英瀚斯生物科技有限公司 | Separation and purification method for HER3 nano antibody |
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