CN104558172A - Single-domain heavy-chain nanobody for amyloid-beta and application of single-domain heavy-chain nanobody - Google Patents
Single-domain heavy-chain nanobody for amyloid-beta and application of single-domain heavy-chain nanobody Download PDFInfo
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- CN104558172A CN104558172A CN201510001584.9A CN201510001584A CN104558172A CN 104558172 A CN104558172 A CN 104558172A CN 201510001584 A CN201510001584 A CN 201510001584A CN 104558172 A CN104558172 A CN 104558172A
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Abstract
The invention discloses a specific single-domain heavy-chain nanobody for amyloid-beta (Abeta), discloses amino acid sequences of a framework region FR and a complementary determining region CDR of a VHH chain of the single-domain heavy-chain nanobody and meanwhile, also discloses a gene sequence for encoding the single-domain heavy-chain nanobody and a host cell capable of expressing the nanobody. By the single-domain heavy-chain nanobody gene sequence and the host cell which are disclosed by the invention, the nanobody can efficiently express in escherichia coli and can be applied to research and development of an amyloid-beta detection reagent and can be used for preparing a medicine for treating the Alzheimer's disease.
Description
Technical field
The present invention relates to biomedicine and biology field, relate to a kind of single domain heavy chain nano antibody for degenerative neural disease virulence factor amyloid.
Background technology
The representative illness alzheimer disease (Alzheimer's disease, AD) of degenerative neural disease, being commonly called as senile dementia, is a kind of persistence neurological dysfunction, is also the origin cause of formation that in elderly population, dementia is the most general.In 2006, about there were 2,600 ten thousand stages alzheimer's disease sufferers in the whole world, was 1/85 to estimating global incidence during the year two thousand fifty.Research display stages alzheimer's disease and brain in old patch (senile plaque, i.e. amyloid plaque) and nerve fiber be entangled with relevant.Current treatment only can help the symptom alleviating disease, the methods for the treatment of of the stages alzheimer's disease course of disease that can not stop or reversing.Till 2012, have how to treat stages alzheimer's disease more than 1000 clinical experimental studies.What comparatively have healing potentiality at present is the various genetic engineering antibody developed for amyloid (amyloid-beta, A β).A beta polypeptides can comprise the amino acid of different number, and common form is as A β 42 and A β 40.These humanized antibody for A β can eliminate it to neuronic toxic action by suppressing the gathering of A β, or reach the effect of replying nervous function by removing established old patch.However, these antibody, due to volume comparatively large (about 150kd), can not readily pass through hemato encephalic barrier, also easily inactivation, sex change, and poor stability, solvability is low and production cost is high, is difficult to apply clinically.
Exist not containing the antibody of light chain in Belgian scientist's reported first camel class blood in 1993, " single domain heavy chain antibody " (VHH) of these disappearance light chains can combine closely with the target such as antigen as normal antibody, and can not stick mutually as conventional antibody, even be gathered into block, complementary reaction can not be caused because of the Fc section containing conventional antibodies in addition.This antibody only comprises a variable region of heavy chain and two conventional CH2 and CH3 districts, the more important thing is that the single domain heavy chain antibody expressed has good structural stability and antigen-binding activity.Molecular weight due to this kind of VHH is 1/10 of common antibody, and size only has 2-5 nm, and therefore VHH also claims nano antibody (Nanobody); Meanwhile nano antibody chemical property is also more flexible, good stability, and solubility is high and easily obtain, and can readily pass through hemato encephalic barrier simultaneously, arrive target site and work.Therefore effectively can overcome conventional antibody shortcoming over the course for the treatment of by the single domain heavy chain nano antibody (called after NB1) filtered out for A β, can be developed into and be used for the treatment of the antibody drug that A β is virulence factor.In addition, because the chemistry of nano antibody, physical stability are good, only containing 130 amino acid of having an appointment, are convenient to multiple marker in genetic engineering modified or chemical coupling, as vitamin H etc., therefore may be used for being developed to the detection kit for A β.
Summary of the invention
Technical problem solved by the invention is: provide a kind of single domain heavy chain nano antibody for A β and aminoacid sequence thereof, the DNA encoding sequence of this single domain heavy chain nano antibody is provided simultaneously, and propose this antibody preparation for A β detection reagent and treatment stages alzheimer's disease medicine in application.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is:
Propose a kind of single domain heavy chain nano antibody for A β, its VHH chain comprises framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, its aminoacid sequence is respectively FR1 shown in SEQ ID NO:1, FR4 shown in the NO:4 of FR3 shown in FR2, SEQ ID NO:3, SEQ ID shown in SEQ ID NO:2; Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, and its aminoacid sequence is respectively the CDR1 shown in SEQ ID NO:5, the CDR3 shown in the CDR2 shown in SEQ ID NO:6, SEQ ID NO:7.
Wherein, the VHH chain of the described single domain heavy chain nano antibody for A β has the aminoacid sequence shown in SEQ ID NO:8.
Also provide a kind of DNA molecular, it is in order to the VHH chain of the above-mentioned single domain heavy chain nano antibody for A β of encoding, and its nucleotide sequence is as shown in SEQ ID NO:9.
In addition, also provide a kind of expression vector, it comprises the nucleotide sequence of above-mentioned DNA molecular, a kind of host cell, and it can express the above-mentioned single domain heavy chain nano antibody for A β.
Present invention also offers single domain heavy chain nano antibody for A β for the preparation of the purposes detected in A β reagent and for the preparation of the purposes in treatment stages alzheimer's disease medicine.
Finally, propose a kind of detection kit for detecting amyloid beta, this test kit comprises the single domain heavy chain nano antibody for amyloid beta recited above.
The invention has the beneficial effects as follows: compared with prior art, advantage of the present invention is as follows:
The present invention adopts A β peptide section immunity Xinjiang two-humped camel, this camel peripheral blood lymphocyte is utilized to establish single domain heavy chain nano antibody gene pool for A β subsequently, A β is coupled on enzyme plate, antigen in this format utilizes display technique of bacteriophage to screen the single domain heavy chain nano antibody gene pool of immunity, thus obtain for A β specific single domain heavy chain nano antibody gene, this gene is proceeded in intestinal bacteria, establishing can in the single domain heavy chain nano antibody strain of E. coli, the gene order of each clone strain is analyzed according to sequence alignment program, obtain the aminoacid sequence for A β single domain heavy chain nano antibody VHH chain, and demonstrate this single domain heavy chain nano antibody can with A β specific binding, thus the exploitation of A β detection reagent and treatment stages alzheimer's disease medicine can be applied to.
Accompanying drawing explanation
Fig. 1 is the bacterium colony PCR electrophorogram constructed phage display single domain heavy chain nano antibody library being carried out to the detection of insertion rate, wherein swimming lane 1 is DNA molecular marker, and swimming lane 2-25 is that the clone PCR of random picking in constructed A β single domain heavy chain nano antibody library detects electrophorogram;
Fig. 2 be express for A β single domain heavy chain nano antibody, the electrophorogram of the SDS-PAGE before and after affinity chromatography purifying, M: protein molecular mark, unit is KDa; 1: before sample is combined with nickel post, the crude extract sample that after broken bacterium, albumen is total; 2: the sample (without band, 15KDa place illustrates that the absorption of single domain heavy chain nano antibody rests on nickel post) after total protein crude extract crosses nickel post; 3-7: totally 5 steps are containing the sample (3-7 successively decrease display take turns basic wash-out completely to the 5th) of 500 mmole imidazole elution wash-outs.
Fig. 3 is the A β detection kit principle schematic based on elisa technique made for core material with A β single domain heavy chain nano antibody NB1.
Reference numeral: the 1. A β single domain heavy chain nano antibody NB1 of bag quilt; 2. antigen A β; 3. A β antibody; 4. enzyme labelled antibody; 5. horseradish peroxidase.
Embodiment
Below in conjunction with Figure of description, the present invention is further illustrated.
The present invention first using the synthetic A β through KLH albumen coupling as antigen immune Xinjiang two-humped camel, after 4 immunity, extract this two-humped camel peripheral blood lymphocyte and construct the special single domain heavy chain antibody library of A β.A β is coupled on enzyme plate, show correct space structure, the epitope of A β is come out, antigen in this format utilizes display technique of bacteriophage to screen the nano antibody gene pool (camel heavy chain antibody phage display gene pool) of A β immunity, and obtain can in the single domain heavy chain nano antibody strain of E. coli.
Below in conjunction with specific embodiment, set forth the present invention further.
Embodiment 1: for the structure in the single domain heavy chain antibody NB1 library of A β
(1) mixed with freund's adjuvant equal-volume by synthesis A β peptide section 20mg (purchased from Nanjing Genscript Biotechnology Co., Ltd.) of KLH albumen coupling, an immunity Xinjiang two-humped camel (purchased from Jurong Sheng Long livestock culturing factory), once in a week, immunity 4 times altogether, except first time uses freund's adjuvant completely, residue all uses not formula Freund's incomplete adjuvant, the specific single domain heavy chain nano antibody VHH for A β of immunologic process moderate stimulation bone-marrow-derived lymphocyte antigen expressed several times.After (2) 4 immunity terminate, extract camel peripheral blood lymphocyte 100ml and extract total serum IgE.(3) the RNA reverse transcription of extraction is become cDNA.(3) utilize sleeve type PCR amplification VHH chain, result such as Fig. 1 shows, and the size of this fragment is about 400 bp.(4) use restrictive restriction endonuclease PstI and NotI enzyme to cut the PCR purified product of 20 μ g pComb3 Vector for Phage Display (Biovector supply) and 10 μ g VHH gene fragments, and connect two fragments.(5) connection product electricity is converted into electricity and turns competent cell TG1(purchased from Divine Land, Beijing red autumnal leaves Science and Technology Ltd.) in, build the single domain heavy chain nano antibody VHH phage display library of A β and measure storage capacity, the size of storage capacity is 6 × 10
8.After library construction completes, be detect the insertion rate in library, what we were random choose 24 clones is bacterium colony PCR(figure mono-).Result shows our insertion rate nearly 100%.
Embodiment 2: the screening process for the single domain heavy chain nano antibody NB1 of A β:
(1) getting 200 u. l transformation TG1 cells joins in 100 milliliters of 2 × TY substratum, cultivates 3 hours for 37 DEG C.(2) add 40 microlitre VCSM13 helper phages, room temperature leaves standstill 30 minutes.(3) centrifugal 10 minutes, by the resuspended access of the cell 250 milliliters of 2 × TY substratum be centrifugation down, 37 DEG C of overnight incubation; (4) by centrifugal for nutrient solution 8000 rpm 30 minutes, get supernatant liquor, with the phage after the amplification of PEG/NaCl precipitation, and the phage after precipitation is dissolved in PBS solution.(5) be coupled on enzyme plate by A β 200 microgram be dissolved in 100 mmole pH 8.2 NaHCO3,4 DEG C of placements are spent the night, and set up negative contrast simultaneously.Add 100 microlitre 0.1% caseins in (26) second days two holes respectively, room temperature closes 2 hours.After (7) 2 hours, add 100 microlitres and adopt the phage that described in above-mentioned steps 4, method is collected, at room temperature act on 1 hour.(8) 0.05% polysorbas20 is contained with in PBST(PBS) wash 5 times, wash away uncombined phage.(9) dissociated down by the phage with A β specific binding with triethylamine (100 mmole), and infect the e. coli tg1 being in logarithmic phase growth, produce also purified phage and be used for the screening of next round, identical screening process repeats 3-4 wheel.In the process of constantly screening, positive clone will constantly by enrichment, thus reaches the object utilizing display technique of bacteriophage sieve to get positive colony.
Embodiment 3: screen the single positive colony of specificity with the enzyme-linked immunoassay method (ELISA) of phage:
(1) contain the Tissue Culture Dish of phage after taking turns screening from above-mentioned 4, select 96 single bacterium colonies and be inoculated in TB substratum containing 100 microgram penbritin every milliliter (containing 2.3 grams of potassium primary phosphates in 1 liter of TB substratum, 12.52 gram dipotassium hydrogen phosphate, 12 grams of peptones, 24 grams of yeast extracts, 4 milliliters of glycerine) in, after growing to logarithmic phase, add the IPTG that final concentration is 1 mmole, 28 DEG C of overnight incubation.(2) utilize osmose process to obtain and slightly carry antibody, and antibody is transferred in antigen coated elisa plate, at room temperature place 1 hour.(3) wash away unconjugated antibody with PBST, add primary antibodie mouse anti-HA tag antibody(mouse-anti HA antibody, purchased from Beijing CoWin Bioscience Co., Ltd.), at room temperature place 1 hour.
(4) wash away unconjugated antibody with PBST, add anti-mouse alkaline phosphatase conjugate(goat-anti-mouse alkaline phosphatase enzyme mark antibody, purchased from the prompt Science and Technology Ltd. of Amy), at room temperature place 1 hour.(5) wash away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, read absorption value at 405 nm wavelength.(6) when sample well OD value is greater than control wells OD value more than 2 times, positive colony hole is judged to.(7) positive colony is seeded to 5 milliliters to contain in the LB liquid of 100 microgram penbritin every milliliter to extract plasmid and to check order.
Analyze the gene order of each clone strain according to sequence alignment program Vector NTI, strain identical for CDR1, CDR2, CDR3 sequence is considered as same clone strain, and the different strain of its sequence is considered as different clone strain, finally has 1 strain antibody.The aminoacid sequence of its antibody is the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 district shown in SEQ ID NO:8, forms whole VHH.
Embodiment 4: anti-A single domain heavy chain nano antibody NB1 is at Host Strains expression in escherichia coli, purifying:
(1) by sequencing analysis above obtain different clone strain plasmid electricity be transformed in intestinal bacteria WK6, and be coated on the plate containing 100 micrograms per millilitre penbritins and 2% glucose LB solid medium, 37 DEG C are spent the night, (2) single colony inoculation is selected in 15 milliliters of LB nutrient solutions containing penbritin, 37 DEG C of shaking table overnight incubation, (3) inoculate 1 milliliter spend the night in bacterial classification to 330 milliliter TB substratum, 37 DEG C of shaking tables are cultivated, when cultivation reaches 0.6-1.0 to OD value, add IPTG, 28 DEG C of shaking table overnight incubation, (4) second days, centrifugal receipts bacterium, (5) osmose process is utilized by thalline to make tropina discharge to obtain antibody crude extract, (6) through affinity chromatography antibody purification albumen, for obtaining highly purified antibody, adopt imidazole gradient elution method, lower concentration imidazole elution (50 mmole) is for washing away assorted band, high density imidazole elution (500 mmole) finally can prepare the albumen that purity reaches more than 90%.Fig. 2 is the electrophorogram of the single domain heavy chain antibody NB1 SDS-PAGE of each step in affinity chromatography purge process for A β expressed; Wherein swimming lane 1 is before being combined with nickel post, the crude extract sample that after broken bacterium, albumen is total, swimming lane 2 is the samples (without band, 15KDa place illustrates that the absorption of this antibody rests on nickel post) after total protein crude extract crosses nickel post, swimming lane 3-7 totally 5 takes turns sample containing 500 mmole imidazole elution wash-outs, and wherein 3-7 successively decreases and illustrates to the 5th and take turns basic wash-out completely.
Embodiment 5: the A β single domain heavy chain nano antibody after purified is for the preparation of the application in the test kit detecting A β content in sample.
As shown in Figure 3, be first coated in ELISA plate by A β single domain heavy chain nano antibody NB1, add the antigen A β standard substance (as 1ng/ml to 1mg/ml) of different gradient concentration, parallel running adds the sample of A β to be detected, at room temperature jog 30 minutes.After washing away unconjugated antigen A β with PBST, add A β single domain heavy chain antibody or other A β antibody again, and for color reaction, the mark that can identify A β single domain heavy chain antibody or other conventional A β antibody two anti-(as the Horseradish Peroxidase Conjugates of rabbit source anti-A β conventional antibody can be identified, the prompt Science and Technology Ltd. of Amy).At room temperature place 1 hour, after washing away unconjugated antibody with PBST, add alkaline phosphatase nitrite ion, on ELISA instrument, at 405nm wavelength, read absorption value.First the absorption value of the A β standard substance of each concentration gradient is made concentration standard curve, then bring the absorption value reading of detected sample into concentration standard curve, the A β content in sample can be judged.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (9)
1. the single domain heavy chain nano antibody for amyloid beta, its VHH chain comprises framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR comprises the aminoacid sequence of FR1, FR2, FR3, FR4, its aminoacid sequence is respectively FR1 shown in SEQ ID NO:1, FR4 shown in the NO:4 of FR3 shown in FR2, SEQ ID NO:3, SEQ ID shown in SEQ ID NO:2; Described complementary determining region CDR comprises the aminoacid sequence of CDR1, CDR2, CDR3, and its aminoacid sequence is respectively the CDR1 shown in SEQ ID NO:5, the CDR3 shown in the CDR2 shown in SEQ ID NO:6, SEQ ID NO:7.
2. the single domain heavy chain nano antibody for amyloid beta according to claim 1, is characterized in that the VHH chain of described single domain heavy chain nano antibody has the aminoacid sequence shown in SEQ ID NO:8.
3. a DNA molecular, is characterized in that, the VHH chain of the single domain heavy chain nano antibody for amyloid beta of its coding described in claim 1 or 2.
4. DNA molecular according to claim 3, is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:9.
5. an expression vector, is characterized in that, it comprises the nucleotide sequence of DNA molecular described in claim 4.
6. a host cell, is characterized in that, it can express the single domain heavy chain nano antibody for amyloid beta described in claim 1.
7. the single domain heavy chain nano antibody for amyloid beta according to claim 1 is for the preparation of the purposes detected in A β reagent.
8. the single domain heavy chain nano antibody for amyloid beta according to claim 1 is for the preparation of the purposes in treatment stages alzheimer's disease medicine.
9., for detecting a detection kit for amyloid beta, it is characterized in that described test kit comprises as claimed in claim 1 for the single domain heavy chain nano antibody of amyloid beta.
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| CN105542005A (en) * | 2016-02-03 | 2016-05-04 | 大连理工大学 | Anti-human amyloid beta-peptide nano antibody and application thereof |
| CN106282214A (en) * | 2016-08-03 | 2017-01-04 | 康众（北京）生物科技有限公司 | A kind of method of quick acquisition nano antibody and application thereof |
| CN112649615A (en) * | 2020-12-21 | 2021-04-13 | 大连理工大学 | Method for detecting soluble Abeta oligomers with different sizes and application |
| CN114578066A (en) * | 2022-05-07 | 2022-06-03 | 北京第一生物化学药业有限公司 | Products and methods for detecting amyloid beta |
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