CN105542005A - Anti-human amyloid beta-peptide nano antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-human amyloid beta-peptide nano antibody (Abeta) and application thereof. A bacteriophage display technique is utilized to carry out multi-screening on an alpaca bacteriophage antibody library to enrich the Abeta-specific bacteriophage; the bacteriophage is cultured to prepare the antibody; identification is carried out to obtain a positive clone; sequencing is carried out to obtain a corresponding coding sequence; and expression is carried out in Escherichia coli to obtain a soluble antibody segment. The antibody disclosed by the invention has the amino acid sequence disclosed as SEQ ID NO.8. The antibody disclosed by the invention has the specific combining capacity with Abeta, and can be used for preparing Abeta-targeted drugs for reagents for disease therapy and diagnosis.

Description

A kind of nano antibody of anti-human amyloid beta and application thereof

Technical field

The invention belongs to Biochemistry and Molecular Biology field, relate to nano antibody and the application thereof of a kind of anti-human amyloid beta (amyloid β, A β).

Background technology

Alzheimer's disease (Alzheimer ' sdisease, AD) be the central nervous system degenerative disease that Progressive symmetric erythrokeratodermia cognition dysfunction and behavior damage as feature.In September, 2015 Alzheimer ' sdiseaseinternational, (ADI) report data display: just have a people to suffer from senile dementia in about every 3.2 seconds at present, in old dementia patients, 60-70% is that Alzheimer characteristic of disease is dull-witted, along with continuing to increase of aged's cardinal sum ratio, AD brings the misery of patient individual and pressure that is social, family will be more and more serious.

Amyloid beta (amyloid β, A β) is the natural product being present in human brain, and it is at neural cell development (HeoC, etal.JNeurochem.2007; 102 (2): 493-500.), sleep regulation (KangJE, etal.2009; 326 (5955): 1005-7), antibacterial (SosciaSJ, etal.PLoSOne2010; 5:e9505.) etc. aspect plays positive regulating effect.But genetic analysis finds A β precursor protein (amyloidprecursorprotein, APP) (GoateA, etal.Nature.1991; 349 (6311): 704-6; JonssonT, etal.Nature.2012; 488 (7409): 96-9) or the gene relevant to its metabolism such as presenilin to undergo mutation (KaminoK, etal.NeurosciLett.1996 relevant with the whether generation of familial AD; 208 (3): 195-8; SherringtonR, etal.Nature.1995; 375 (6534): 754-60.), therefore it is believed that A β is certainly relevant to the generation of AD.In AD generating process, free A β experience first increases the process (MaiaLF, the etal.EMBOMolMed.2015 that reduce afterwards; 7 (7): 895-903.), may just because of the metabolic imbalance of A β, A β assembles the solvable A beta peptide aggregation body of formation and insoluble A beta deposition gradually under the induction of certain unknown condition.

Research of Animal Model for Study and clinical experiment all find that common antibody can disturb the metabolism of A β to a certain extent, but there is no desirable result in alleviation AD PD.Carefully analyze therapeutic process to find, its cure mechanism mainly breaks the balance of blood and cerebrospinal fluid A β by the combination of A β in medicine and blood, impels A β in cerebrospinal fluid to cross over hemato encephalic barrier and enters blood; Or play direct result for the treatment of by antibody leap hemato encephalic barrier, if but actual drug main IgG antibody-like used, A β level extremely low in blood and antibody are difficult to the defect of crossing over hemato encephalic barrier, greatly limit its result for the treatment of (BardF, etal.ExpNeurol.2012; 238 (1): 38-43.).

Cross over the ability of hemato encephalic barrier to improve antibody, antagonist carries out embedding and modifies or target antibody and the albumen (cerebrovascular endothelial cell associated antigen or have the albumen of special transcytosis) had across hemato encephalic barrier ability are built into mosaic usually at present.No matter any strategy, all further increases the preparation cost of antibody.And the nano antibody from cameloid antibody heavy chain variable region of molecular weight (12-15kD) iso-electric point higher (pI>9.2) can cross over hemato encephalic barrier (LiT, etal.FASEBJ.2012 very efficiently under the condition of not carrying out any modification; 26 (10): 3969-79.), therefore nano antibody has good application prospect in disease of brain treatment; In addition, the nano antibody of acquisition equally with common antibody may be used for the A β diagnosis that is target, detects and treat.

Summary of the invention

An object of the present invention is to provide a kind of nano antibody of anti-amyloid beta.

A kind of nano antibody of anti-amyloid beta, described nano antibody aminoacid sequence comprises framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR is made up of the aminoacid sequence of the FR of lower group: the FR1 as shown in SEQIDNO.1, FR2 as shown in SEQIDNO.2, FR3 as shown in SEQIDNO.3, the FR4 as shown in SEQIDNO.4; Described complementary determining region CDR is made up of the aminoacid sequence of the CDR of lower group: the CDR1 as shown in SEQIDNO.5, the CDR2 as shown in SEQIDNO.6, the CDR3 as shown in SEQIDNO.7.

Further, the nano antibody of described anti-amyloid beta, it has the aminoacid sequence shown in SEQIDNO.8.

Two of the object of the invention is to provide the application of a kind of nano antibody described above in the medicine preparing the disease that treatment amyloid beta metabolic imbalance causes.Preferably, the disease that described amyloid beta metabolic imbalance causes is alzheimer's disease.

Three of the object of the invention is to provide the application of a kind of nano antibody described above in the detection reagent of preparation detection amyloid beta.

The present invention also provides a kind of DNA molecular, the nano antibody of its anti-amyloid beta of the present invention of encoding.

Beneficial effect of the present invention:

The invention provides the nano antibody of a species specific anti-A β, this nano antibody has the aminoacid sequence different from delivering antibody to A β, the gene of its correspondence can be transferred to protokaryon or express in eukaryotic expression system, the antibody of acquisition can be applied in various with in the A β preparation of medicine of disease that is target and the preparation of the detection reagent of A β.

Accompanying drawing explanation

Fig. 1 is the framework region of nano antibody of the present invention, the position of complementary determining region and aminoacid sequence schematic diagram.

Fig. 2 is the expression of SDS-PAGE electrophoresis detection nano antibody under IPTG inductive condition.M is protein molecular weight standard; Swimming lane 3 is the whole bacterial protein not adding IPTG induction; Swimming lane 1 and 2 for IPTG induce after whole bacterial protein.

Fig. 3 is SDS-PAGE electrophoresis detection metal chelate chromatography separation and purification nano antibody situation.M is protein molecular weight standard, and swimming lane 1 wears liquid for stream; Swimming lane 2-4 is 50mM imidazoles wash-out; Swimming lane 5-11 is 150mM imidazoles wash-out; Swimming lane 12-13 is 500mM imidazoles wash-out.

Fig. 4 is that ELISA detects nano antibody to different sequence (A β _12-35, A β ₄₀, A β ₄₂) with the binding ability of state (monomer, can insoluble aggregate and fiber) A β.The difference of control group and experimental group is that the former envelope antigen does not add nano antibody, and the latter adds nano antibody.

Fig. 5 is that MTT detects nano antibody to the protection situation of A β process cell SH-SY5Y.

Embodiment

Following non-limiting example can make those skilled in the art understand the present invention more comprehensively, and DNA sequence dna disclosed in the present application and aminoacid sequence can obtain according to the additive method that this area is conventional.And corresponding gene constructed to any suitable carrier of the new aminoacid sequence that the application can be obtained, express with any suitable expression system.Following content is only the exemplary illustration to the scope that this application claims protection, and some changes made according to disclosed content and modification, also should belong to the scope that this application claims protection.If the medicine used in embodiment herein and reagent are without specified otherwise approach acquisition all routinely.

Not homotactic A β (A β _12-35, A β ₄₀, A β ₄₂) monomer, can insoluble aggregate and fiber purchased from Zhongtai Bio-Chem. Co., Ltd., Hangzhou.

The foundation in embodiment 1 nano antibody storehouse

The nonimmune storehouse that the phage display library that the present invention uses is is carrier with T7 phage, establishment step is as follows:

(1) extract 2 years old male alpaca peripheral blood lymphocyte total serum IgE ( puerLinkTMRNAMiniKit, LifeTechnologies:12183018A); Take UPprimer1 as primer by the total serum IgE reverse transcription of extracting be cDNA, and adopt two-wheeled nested PCR amplification VHH gene; Wherein first round PCR is the cDNA transcribed is masterplate, with UPprimer1 and DOWNprimer1 for primer, the band that size is 650 ~ 750bp is reclaimed after pcr amplification, again as masterplate, carry out second and take turns pcr amplification, upstream and downstream primer is respectively UPprimer2 and DOWNprimer2, reclaims the PCR primer obtaining 450 ~ 500bp;

UPprimer1:5’-GGTACGTGCTGTTGAACTGTTCC-3’

DOWNprimer1:5’-CTTGGTGGTCCTGGCTGCTCT-3’

UPprimer2:5’-AAGCTTTTGTGGTTTTGGTGTCTTGGGTTC-3’

DOWNprimer2：5’-AAGCTTGGGGTCTTCGCTGTGGTGCG-3’

(2) cut above-mentioned PCR primer with EcoRI and HindIII enzyme, be VHH gene fragment;

(3) with T4 ligase enzyme connect T7 carrier ( 10-3CloningKit, MerckMillipore 70550-3) with VHH gene fragment, connection product is packed, then carries out increase (method is shown in embodiment 2), obtain phage primary libraries.

The liquid amplification of embodiment 2T7 phage

(1) activate Host Strains: inoculation intestinal bacteria BLT5403 in 50mlLB substratum (containing 0.1mg/mL Pyocianil, being called for short Carb-LB), 37 DEG C of 170r/min incubated overnight, obtain generation Host Strains;

(2) in Carb-LB liquid nutrient medium, inoculate the generation Host Strains of 1%, shaking table is cultivated, to OD ₆₀₀for 0.5-1.0, obtain two generation Host Strains;

(3) with T7 phage-infect two generation Host Strains, 37 DEG C of 200r/min cultivate 2-3h, observe bacteriolyze situation, and bacteriolyze stops cultivating completely immediately.By lysate at the centrifugal 10min of 8000g, and reclaim supernatant, supernatant is the phage of amplification.Measure titre.

The mensuration of embodiment 3T7 phage titre

(1) in culture dish, pour Carb-LB bottom substratum into, cool, solidify;

(2) dissolve top layer substratum, cooling, add 45 DEG C of insulations after Carb;

(3) with the phage of aseptic LB liquid nutrient medium gradient dilution amplification;

(4) 300 μ L bis-generation Host Strains mix from the phage of the different gradient dilution of 50 μ L, add 4mL preheating Carb-top layer substratum, pour into rapidly in the plate of bottom substratum, rock plate and make it to be evenly distributed;

Be inverted for (5) 37 DEG C and cultivate 3-4h, until see the plaque be of moderate size;

(6) selecting plaque quantity is that flat board between 50-500 carries out plaque count, calculates phage titre.

The screening of embodiment 4 nano antibody

(1) A β TBS is diluted to 10 μ g/mL, adjust pH to 7.0, enzyme plate bag quilt is carried out in 100 μ L/ holes;

(2) plate is washed 3 times with TBS after room temperature shaker reaction 2h;

(3) close without protein blocking liquid (purchased from Sheng Gong biotechnology company limited) with 1%, after room temperature shaker 1.5h, wash plate 6 times with TBST;

(4) add the phage of amplification, 100 μ L/ holes, after room temperature shaker hatches 30min, wash plate 10 times with TBST, fully remove unconjugated phage;

(5) add T7 elution buffer (1%SDS) wash-out bacteriophage, room temperature shaker 30min, and elutriant is increased, obtain next round screening with phage, enter next round screening;

(6) after four-wheel screening, specific phage obtains enrichment.

The sequential analysis of embodiment 5 monoclonal phage

After fourth round screening, obtain mono-clonal plaque according to the method for embodiment 3.PCR method increases laggard row sequential analysis, and method is as follows:

(1) pcr amplification

1. use sterilizing toothpick picking plaque, a same plaque part puts into EDTA colloidal sol damping fluid for PCR, and another part increases, for follow-up ELISA;

2. the part 65 DEG C process 10min of EDTA colloidal sol damping fluid is put into;

3. be cooled to room temperature, the centrifugal 5min of 14000g, get supernatant 98 DEG C of thermal shock process 5min;

4. get 2 μ L and carry out pcr amplification as template;

Reaction conditions is: after 94 DEG C of denaturation 5min, 98 DEG C of sex change 10s, 55 DEG C of annealing 30s, and 72 DEG C extend 60s, 30 circulations, and last 72 DEG C extend 7min;

(2) PCR primer is delivered Hua Da gene sequencing;

(3) nucleotide sequence obtained be converted into aminoacid sequence and compare, its aminoacid sequence is SEQIDNO.8, is made up of 4 framework regions (FR), 3 complementary determining regions (CDR).Aminoacid sequence and its composition sequence of described nano antibody are as follows.

Aminoacid sequence is as shown in SEQIDNO.8, and wherein the aminoacid sequence of 4 framework regions FR1, FR2, FR3 and FR4 respectively is shown in SEQIDNO.1 ~ 4; 3 complementary determining region CDR1, CDR2, CDR3 aminoacid sequences respectively are shown in SEQIDNO.5 ~ 7; Position relationship is as shown in Figure 1 in SEQIDNO.8 for described framework region, complementary determining region.

The expression of embodiment 6A β nano antibody and separation and purification

(1) expression of nano antibody:

1. the gene order (as SEQIDNO.9) of nano antibody is inserted expression vector pET23a, and be transformed into e. coli bl21 (DE3) cell;

2. use LB substratum, cultivate under the condition of temperature 37 DEG C, treat thalline OD ₆₀₀when reaching 1-1.5, adding final concentration is that the IPTG of 1mmol induces;

3. continue to cultivate after 3-4 hour and abandon supernatant, collect full bacterium;

4. use ultrasonic degradation cell after the resuspended thalline of lysis buffer, at the centrifugal 5min of 10000rpm, collect supernatant liquor and precipitation.With the expression of 15%SDS-PAGE electrophoresis detection nano antibody, result as shown in Figure 2.

In Fig. 2, M is protein molecular weight standard; Swimming lane 3 is the whole bacterial protein not adding IPTG induction; Swimming lane 1 and 2 for IPTG induce after whole bacterial protein, have the protein expression spot of obvious nano antibody of the present invention at 16.5kDa place.Relatively swimming lane 3 and swimming lane 1 or 2 find, after adding IPTG, nano antibody expression amount increases.

(2) purifying of nano antibody: adopt Ni-NTA affinity column purified fusion protein:

1. chromatography column is with after sample-loading buffer (15mmolPBS, 50mmol imidazoles, 500mmolNaCl) cleaning, adds the supernatant liquor collected in above-mentioned (1);

2. first use cleaning buffer solution (15mmolPBS, 150mmol imidazoles, 500mmolNaCl) to clean and remove foreign protein, then use elution buffer (15mmolPBS, 500mmol imidazoles, 500mmolNaCl) wash-out and collect elutriant;

3. after purifying, nano antibody is with the purifying situation of 15%SDS-PAGE electrophoresis detection fusion rotein, and result as shown in Figure 3.

SDS-PAGE electrophoresis detection metal chelate chromatography separation and purification nano antibody situation in Fig. 3.M is protein molecular weight standard, and swimming lane 1 wears liquid for stream; Swimming lane 2-4 is 50mM imidazoles wash-out; Swimming lane 5-11 is 150mM imidazoles wash-out; Swimming lane 12-13 is 500mM imidazoles wash-out.The result display of Fig. 3, nano antibody is after affinity chromatography process, and obtain the nano antibody that band is comparatively single, its molecular weight is about 16.5kDa.

Embodiment 7ELISA detects the binding specificity of nano antibody

(1) the A β (0.5mg/L) of different for 100 μ l sequence and type is joined on elisa plate, after 37 DEG C of incubation 1h, place in 4 DEG C of refrigerators and spend the night;

(2) use up liquid in plate hole, fill it up with PBST washings (NaCl8g, KH ₂pO ₄0.2g, Na ₂hPO ₄12H ₂o2.9g, Tween-200.5ml, be settled to 1000ml with distilled water), leave standstill 3-5min, 3 times repeatedly, finally plate is upside down on thieving paper, washings in hole is flow to end;

(3) add confining liquid (0.5% bovine serum albumin, the PBS of pH7.4) 200 μ l, wash after 37 DEG C of placement 1h, wash same step (2);

(4) every hole adds nano antibody 200 μ l, washs, wash same step (2) after 1 hour;

(5) diluted by 1:1000 by anti-6 × HisTagantibody (0.5mg/ml), every hole adds 100 μ l, washs, wash same step (2) after 37 DEG C of placement 1h;

(6) diluted by 1:1000 by goat anti-rabbit igg-HRP (0.5mg/ml), every hole adds 200 μ l, places 1h for 37 DEG C;

(7) add TMB working fluid 200 μ l, under room temperature, in the dark place 10-15min, add stop buffer, every hole 50 μ l;

(8) with the reading at microplate reader record 450nm place, result as shown in Figure 4;

In Fig. 4, nano antibody is to the A β of different state of aggregation (monomer, can insoluble aggregate and fiber) and not homotactic A β (A β _12-35, A β ₄₀, A β ₄₂) there is binding ability.

Embodiment 8MTT detects antibody to the provide protection of A β process cell

(1) Human neuroblastoma cell SH-SY5Y is with 10 ⁶individual/ml is inoculated in 25cm ²tissue Culture Flask, nutrient solution is for containing 10% foetal calf serum (NQBB, Australia) and 1% dual anti-(ThermoFisherScientific, USA) DMEM high glucose medium (Gibco, USA), culture condition is 37 DEG C, 5% carbonic acid gas;

(2) go down to posterity with 0.25% tryptic digestion after cell reaches 80% degree of converging, the cell in vegetative period of taking the logarithm carries out bed board with 5000/hole, continue in cell culture incubator and cultivate 24h to make cell attachment;

(3) after cell attachment, every aperture is crossed PBS and is washed 1 time, is replaced by 100 μ LDMEM high glucose medium/N2 additives (Gibco, USA), continues to cultivate 1h;

(4) every hole adds final concentration is the A beta peptide aggregation body of 15 μMs or the nano antibody (nanobody) of A beta peptide aggregation body and different concns, continues to cultivate 48h;

(5) by nutrient solution sucking-off, wash once with PBS, every hole adds 90 μ LDMEM high glucose mediums and 10 μ L5mg/mlMTT (Amresco, USA) solution, is positioned in cell culture incubator and continues to cultivate 4h;

(6) nutrient solution in the careful every hole of sucking-off, adds 150 μ LDMSO, and level concussion makes DMSO fully dissolve the crystallization of bluish voilet Jia Za, and measure 570nm absorbancy, reference wavelength is 630nm.Result as shown in Figure 5.

In Fig. 5, nano antibody not only can reverse the cell injury that A beta peptide aggregation body causes, and does not have cytotoxicity to SH-SY5Y, and can also promote the increment of SH-SY5Y to a certain extent, and above-mentioned effect has dose-dependently.

Claims (5)

1. the nano antibody of an anti-amyloid beta, described nano antibody aminoacid sequence comprises framework region FR and complementary determining region CDR, it is characterized in that, described framework region FR is made up of the aminoacid sequence of the FR of lower group: the FR1 as shown in SEQIDNO.1, FR2 as shown in SEQIDNO.2, FR3 as shown in SEQIDNO.3, the FR4 as shown in SEQIDNO.4; Described complementary determining region CDR is made up of the aminoacid sequence of the CDR of lower group: the CDR1 as shown in SEQIDNO.5, the CDR2 as shown in SEQIDNO.6, the CDR3 as shown in SEQIDNO.7.

2. the nano antibody of anti-amyloid beta according to claim 1, is characterized in that, it has the aminoacid sequence shown in SEQIDNO.8.

3. the nano antibody described in claim 1 or 2 treats the application in the medicine of the disease that amyloid beta metabolic imbalance causes in preparation.

4. application according to claim 3, is characterized in that, the disease that described amyloid beta metabolic imbalance causes is alzheimer's disease.

5. the nano antibody described in claim 1 or 2 detects the application in the detection reagent of amyloid beta in preparation.