CN1635119A - Process for preparing amyloidosis polypeptide beta (Abeta), a senile dementia causing substance, and product yield thereof - Google Patents

Abstract

The invention provides a method for mass-production of senile dementia nosogenetic substance Abeta polypeptide characterized in fusing the bacterium Intein onto the amidogen end or carboxyl end of the Abeta polypeptide. to make the short peptides which are difficult to express in bacteria express diffusely in bacteria. The invention also relates to a fusion gene for fusing the Interin-Abeta protein, and a prokaryocyte expression carrier containing the gene. The invention also relates to the bacteria expression and purification of the Interin-Abeta protein, and hydrolysis release and purification method of the Abeta peptide from the fusion protein.

Description

Method and the product thereof of a kind of production senile dementia morbid substance amyloidosis polypeptide β (A β)

Technical field

The required polypeptide expression of relate generally to treatment senile dementia of the present invention more specifically the invention provides a kind of core composition that can treat and prevent the vaccine of senile dementia.

Background technology

Senile dementia (mainly refer to alzheimer's disease＜Alzheimer ' s Disease, AD＞) sickness rate is in rising trend along with social population's aging.The AD patient who suffers from the total population more than 200,000,000 in various degree in the U.S. just has 4,800,000, accounts for 2% of total population, and sickness rate is higher in the old man more than 60 years old.This disease has the gesture of the prevailing disease of being development in recent years, and following sickness rate also can be higher.In China akin sickness rate is arranged also.The main pathological characters of AD disease is to produce the neuropathy patch with memory and cognitive relevant zone in patient's brain, main component is the polypeptide of a kind of molecular weight 4KD in the patch, the Amyloid that beta amyloid peptide (A β) is polymerized and getting involved, and some inflammatory reaction neurogliocyte in wherein the malnutrition or the neural dendron and the aixs cylinder of sex change.Also produce a large amount of neurofibrillary tangleses in the most of patients patch.The study of pathogenesis of AD disease shows that A β and fibroplastic Amyloid thereof are main pathologies, because a large amount of Amyloids deposition and the inflammatory reactions in cerebral diseased district, makes neurocyte atrophy, sex change, the defunctionalization at this place.Cause the impaired symptom of a series of neural functions to occur.Therefore, control AD disease should mainly be carried out from two aspects: a large amount of generations, accumulation and the formation fiber that promptly stop the A beta polypeptides; And remove A beta polypeptides, fiber and the Amyloid that the cerebral diseased district has produced.

A direction of the drug development of AD disease is immunotherapy at present.One of method is to use the synthetic starch sample to become polypeptide A β and produces anti-amyloid beta antibodies as the boosting vaccine body, thereby accelerates the interior immunocyte of brain with the removing of Fibrotic A β from cerebral tissue.This vaccine is effective (the Schenk D 1999 of proof in the mouse animal experiment; Morgan D 2000; Weiner H2000; Janus C 2000; Lemere C 2001; Poduslo JF 2001).But, use synthetic polypeptide as vaccine cost height, clinical application is restricted.Bacterial expression albumen, peptide chain are short more, and hydrophobicity is strong more, express difficult more.A β is a small peptide that hydrophobicity is very strong, directly with this small peptide of bacterial expression very big difficulty is arranged.Given this, express difficulty for overcoming hydrophobic small peptide, inventor's imagination is utilized gene engineering method that A beta polypeptides gene is merged the back with the hydrophilic protein gene that is easy to express and is expressed, and can improve the expression productive rate.Through bacterium great expression and preliminary purification, with the albumen excision of merging, be further purified target polypeptides-A beta polypeptides again.According to this thinking, the inventor has carried out deep research, has finished the present invention finally.

Summary of the invention

One aspect of the present invention relates to a kind of fusion rotein, and its Intein albumen by A beta polypeptides and bacterium is formed, and described A beta polypeptides is blended in carboxyl terminal and/or the aminoterminal of Intein.A beta polypeptides aminoacid sequence is to be derived by people A beta polypeptides sequence, comprises 40 or 42 amino acid whose A beta polypeptides sequences.

Another aspect of the present invention relates to a kind of fusion gene, its A beta fusion proteins of the present invention of encoding.

The prokaryotic vector that comprises described fusion gene that relates in one aspect to again of the present invention, and transform or the prokaryotic host cell of transfection by this expression vector.

Another aspect of the present invention relates to the method for producing the A beta polypeptides, and this method is included in the fusion rotein of expressing Intein-A β in the prokaryotic cell prokaryocyte, and the A beta polypeptides is discharged from this fusion rotein hydrolysis, and purifying obtains the A beta polypeptides.

The present invention can realize in the following way:

A β-Intein the fusion rotein of bacterial expression production of the present invention has multiple mode.A kind of is to add a methionine(Met) before the A beta polypeptides, and its carboxyl terminal and Intein2 albumen merge; Another kind merges the aminoterminal of Intein1 protein D naB and A beta polypeptides; The third uses two Intein albumen and A β to merge, and the aminoterminal that DnaB is blended in A β merges; Intein2 (GyrA or RIR1) merges with the carboxyl terminal of A β.

Hold the bacterial expression vector of germy expression promotor all to can be used for realizing the present invention at 5 ' of A β and Intein fusion gene.The used expression vector of the present invention is Twin1 (New EnglandBiolabs), and as shown in Figure 1, it has the T7 promotor, carries N and C-terminal simultaneously and merges required Intein, so that select N or/and C-terminal and Intein albumen merge when vector construction.

The constructed A β-Intein fusion rotein of the present invention only contains an Intein; (Fig. 2 a) in the N-terminal fusion of proteic DnaB of Intein and A β; Or the C-terminal of proteic GyrA of Intein and A β merges (Fig. 2 b).

A beta polypeptides coding region has following one of them aminoacid sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(Aβ42)

Or DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (A β 40)

Or have continuous 5 amino acid whose sequences in above-mentioned arbitrary aminoacid sequence.

Adopt in above-mentioned A beta polypeptides or its aminoacid sequence continuous 5 amino acid whose polypeptide all can stimulate body immune system to produce immune antibody as the immunizing antigen vaccine.But small peptide is very low at the expression efficiency of bacterium.

For obtaining above-mentioned A beta polypeptides, can select for use several schemes that A beta polypeptides and Intein albumen are merged.

(1) aminoterminal of Intein1 protein D naB and A beta polypeptides merges

The dna direct of coding A beta polypeptides is added in the proteic DnaB carboxyl terminal of vector encoded Intein1, removes the Intein2 Protein G yrA on the Twin1 carrier.Thereby obtain the DNA construct of the A beta polypeptides of coding aminoterminal and Intein albumen fusion.The structure of the DnaB-A beta polypeptides that the present invention is selected is:

MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSN

VPALWQLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEIWAI

NEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDE

LSLKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGP

HNFVANDIIVHN DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

It is A β 40 polypeptide that the carboxyl terminal that rolls off the production line is wherein arranged.The enzymolysis site of Intein1 is at the proteic carboxyl terminal of Intein1, so complete A beta polypeptides can be discharged behind the enzymolysis.The structural dna sequence of this fusion rotein of encoding is:

CATATGAAAATCGAAGAAGGTAAACTGACAAATCCTGGTGTATCCGCTTGG

CAGGTCAACACAGCTTATACTGCGGGACAATTGGTCACATATAACGGCAAG

ACGTATAAATGTTTGCAGCCCCACACCTCCTTGGCAGGATGGGAACCATCC

AACGTTCCTGCCTTGTGGCAGCTTCAAAACAACGGTAACAACGGTCTCGA

ACTGCGCGAGTCCGGAGCTATCTCTGGCGATAGTCTGATCAGCCTGGCTAG

CACAGGAAAAAGAGTTTCTATTAAAGATTTGTTAGATGAAAAAGATTTTGA

AATATGGGCAATTAATGAACAGACGATGAAGCTAGAATCAGCTAAAGTTAG

TCGTGTATTTTGTACTGGCAAAAAGCTAGTTTATATTCTAAAAACTCGACTA

GGTAGAACTATCAAGGCAACAGCAAATCATAGATTTTAACTATTGATGGTT

GGAAAAGATTAGATGAGCTATCTTTAAAAGAGCATATTGCTCTACCCCGTAA

ACTAGAAAGCTCCTCTTTACAATTGTCACCAGAAATAGAAAAGTTGTCTCA

GAGTGATATTTACTGGGACTCCATCGTTTCTATTACGGAGACTGGAGTCGAA

GAGGTTTTTGATTTGACTGTGCCAGGACCACATAACTTTGTCGCGAATGAC

ATCATTGTACACAACGATGCAGAATTCCGACATGACTCAGGATATGAAGTC

CATCATCAAAAATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAAAGGT

GCAATCATTGGACTCATGGTGGGCGGTGTTGTCTAACTGCAG

Wherein 5 ' end has and rolls off the production line CATATGSequence is a restriction endonuclease NdeI recognition site, 3 ' end CTGCAGSequence is a restriction endonuclease PstI recognition site.It is different with common restriction endonuclease wherein to clone used SapI restriction endonuclease, and restriction enzyme site is outside recognition site, and the SapI site has not existed to cause DNA to make up afterwards.The advantage of using the SapI restriction endonuclease is that the junction at fusion rotein can not import extra aminoacid sequence because of keeping restriction enzyme site.The construction process of this structure is fairly simple, and A β 40 gene clones are advanced between the SapI of Twin1 expression vector and the PstI restriction enzyme site to get final product.The carrier structure that contains DnaB-A β 40 fusion roteins is asked for an interview Fig. 2 a.

A β 40 in the fusion rotein separates, purifying can use Chitin Bead (New EnglandBiolabs) affinity column to be adsorbed in Intein-A β 40 fusion roteins of bacterial expression on the affinity column in ealkaline buffer, then the pH value is transferred to neutrality, at position of fusion A β 40 hydrolysis are discharged at 25 degree room temperature Intein protein D naB.

(2) add a methionine(Met) before the A beta polypeptides, its carboxyl terminal and Intein2 albumen merge

The carboxyl terminal of the DNA of coding A β 40 polypeptide is merged with the proteic encoding gene of Intein2, and with the removal of the Intein1 protein D naB coding region on the carrier, add an albumen initial amino acid-methionine(Met) at the aminoterminal of the encoding sequence of A β 40.The selected Intein2 albumen of the present invention is the entrained GyrA of Twin1 carrier.The peptide sequence structure of A β 40-GyrA fusion rotein is:

M DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVCITGDALVALP

EGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTV

EGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFA

RGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVT

DAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTA

YTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ

It is A β 40 polypeptide that the amino terminal sequence that rolls off the production line is wherein arranged.The enzymolysis site of Intein2 is at the proteic aminoterminal of Intein2, so can discharge the A beta polypeptides that has extra methionine(Met) behind the enzymolysis.The structural dna sequence of this fusion rotein of encoding is:

CATATGGATGCAGAATTCCGACATGACTCAGGATATGAAGTCCATCATCAAAA

ATTGGTGTTCTTTGCAGAAGATGTGGGTTCAAACAAAGGTGCAATCATTGGA

CTCATGGTGGGCGGTGTTGTCTGCATCACGGGAGATGCACTAGTTGCCCTAC

CCGAGGGCGAGTCGGTACGCATCGCCGACATCGTGCCGGGTGCGCGGCCCA

ACAGTGACAACGCCATCGACCTGAAAGTCCTTGACCGGCATGGCAATCCCG

TGCTCGCCGACCGGCTGTTCCACTCCGGCGAGCATCCGGTGTACACGGTGCG

TACGGTCGAAGGTCTGCGTGTGACGGGCACCGCGAACCACCCGTTGTTGTG

TTTGGTCGACGTCGCCGGGGTGCCGACCCTGCTGTGGAAGCTGATCGACGA

AATCAAGCCGGGCGATTACGCGGTGATTCAACGCAGCGCATTCAGCGTCGAC

TGTGCAGGTTTTGCCCGCGGAAAACCCGAATTTGCGCCCACAACCTACACA

GTCGGCGTCCCTGGACTGGTGCGTTTCTTGGAAGCACACCACCGAGACCCG

GACGCCCAAGCTATCGCCGACGAGCTGACCGACGGGCGGTTCTACTACGCG

AAAGTCGCCAGTGTCACCGACGCCGGCGTGCAGCCGGTGTATAGCCTTCGT

GTCGACACGGCAGACCACGCGTTTATCACGAACGGGTTCGTCAGCCACGCT

ACTGGCCTCACCGGTCTGAACTCAGGCCTCACGACAAATCCTGGTGTATCCG

CTTGGCAGGTCAACACAGCTTATACTGCGGGACAATTGGTCACATATAACGG

CAAGACGTATAAATGTTTGCAGCCCCACACCTCCTTGGCAGGATGGGAACCA

TCCAACGTTCCTGCCTTGTGGCAGCTTCAATGA CTGCAG

Wherein 5 ' end has and rolls off the production line CATATGSequence is a restriction endonuclease NdeI recognition site, 3 ' end CTGCAGSequence is a restriction endonuclease PstI recognition site.Similar to aforementioned aminoterminal fusion method, its SapI restriction enzyme site disappears in the DNA building process.The construction process of this structure is fairly simple, and A β 40 gene clones are advanced between the NdeI of Twin1 expression vector and the SapI restriction enzyme site to get final product.The carrier structure that contains A β 40-GyrA fusion rotein is asked for an interview Fig. 2 b.

A β 40 in the fusion rotein separates, purifying uses the Chitin affinity column that Intein-A β 40 fusion roteins of bacterial expression are adsorbed on the affinity column equally, use reductive agent then, for example DTT induces Intein2 Protein G yrA at position of fusion A β 40 hydrolysis to be discharged.

(3) two Intein albumen and A β merge

A β 40 polypeptide and two Intein albumen are merged; A β 40 carboxyl terminales and Intein2 albumen are merged, A β 40 aminoterminals and Intein1 protein D naB are merged.The bacterial expression of fusion method and fusion rotein and purification process are similar to aforesaid method.The method that hydrolysis discharges A β 40 need be used in combination reductive agent and adjust pH and method of temperature.

The method that makes up A β 42-Intein fusion rotein is similar to above-mentioned A β 40-Intein.Difference is to replace A β 40 construction of fusion protein expression vectors with A β 42 peptide sequences.A β 42-Intein Expression of Fusion Protein, purifying and hydrolysis discharge then identical with A β 40-Intein fusion rotein.

Certainly, other known Intein protein sequence, or other bacterioprotein also can be used as the fusion object.Here cited dna sequence dna wherein has 1st/3rd at least, can change and does not influence the peptide sequence and the protein structure of expressed fusion rotein.

Beneficial effect

The present invention expresses by A beta polypeptides gene is merged the back with the hydrophilic protein gene that is easy to express, and has improved the expression productive rate.Through bacterium great expression and preliminary purification, with the albumen excision of merging, be further purified target polypeptides-A beta polypeptides again.

The accompanying drawing summary

Fig. 1 is a TWIN-1 carrier structure synoptic diagram.

Fig. 2 a. is to be that the carrier synoptic diagram that the back makes up is merged A β 40 polypeptide N-terminal DNA and InteinDNA with in the basis with TWIN-1.

Fig. 2 b. is to be that the carrier synoptic diagram that the back makes up will be merged at terminal DNA of A β 40 peptide Cs and InteinDNA in the basis with TWIN-1.

The electrophorogram of Fig. 3 a.TWIN1-N-A β 40 abduction delivering in bacterial strain.1 is molecular weight standard (kD); 2 inductive bacterial strains not; Expression strain after 3 process IPTG induce; The supernatant liquor of 4 inducible strains behind the broken bacterium of ultrasonic wave; 5,7 be the inclusion body of inducible strain behind the broken bacterium of ultrasonic wave; 6 are the inclusion body after the washing.

Electrophorogram after Fig. 3 b.TWIN1-N-A β 40 expression products are purified.1,2,3 is A β 40 expression products of inclusion body behind the ChitinBeads affinitive layer purification; 4 is molecular weight standard (kD).

The description of embodiment

Embodiment 1:A β 40 aminoterminals merge the bacterial expression of the fusion rotein of Intein1

One, makes up the DNA of coding Intein1-A β 40 fusion roteins

Use PCR method to become precursor protein (Amyloid precursorprotein (APP)) selective amplification A β 40 polypeptide genes from human amyloid.Select the EXPEND HIGH FIDELITY PCR KIT of ROCHE company for use.The PCR reaction comprises 38ul deionized water, the 10X damping fluid that the 5ul test kit provides, 3ul DMSO, 1uldNTP (10mM dATP, 10mM dCTP, 10mM dGTP, 10mM dTTP), the hybrid dna polysaccharase that the 1ul test kit provides, 1ul 10ng/ml contain the DNA plasmid of people APP as template.1ul 10pM forward primer and 1ul 10pM inverse pairs primer.

Select the forward primer A β-N-SapF that contains the Sap1 restriction enzyme site for use:

GATCTCA GCTCTTCTAACGATGCAGAATTCCGACATG; Select the reverse primer A β 40-N-Pst-R that contains the Pst1 restriction enzyme site for use:

GTGCATTGGTT CTGCAGTTAGACAACACCGCCCACCATG A β 40 polypeptide) or A β 42-N-pst-R:CACTATG CTGCAGCTACGCTATGACAACACCGCC (A β 42 polypeptide).The primer combinations of pairs of PCR reaction is summarized as follows:

Aβ40：Aβ-N-SapF+Aβ40-N-Pst-R

Aβ42：Aβ-N-SapF+Aβ42-N-pst-R

PCR reaction is used 94 ℃, 30 seconds 54 ℃, 30 seconds 72 ℃, 1 minute; Totally 30 loop cycles.Extract 2ul PCR product and add 3ul deionized water and 1ul electrophoresis solution (SigmaG2526), after the separation of 1% agar electrophoresis confirms that the PCR product contains the DNA of target gene size, all the other PCR products are used the QIAQUIK PCRPURIFICATION KIT purifying of QIAGEN company: a PCR reaction product is mixed with five portions of PB damping fluids that test kit provided, and the small-sized centrifugal post that test kit provided of packing into then is centrifugal; Re-use that 0.5ml PB damping fluid is centrifugal washes post once; The EB damping fluid that is provided with the 50ul test kit is adorned the centrifugal wash-out of post again.Through the DNA concentration determination, draw the PCR product of 1ug purifying, add 2ul 10X damping fluid, 1ul SapI restriction endonuclease and 1ul PstI restriction endonuclease (New EnglandBiolabs) add deionized water again to final volume 20ul, and 37 ℃ are incubated 2 hours.1ugTwin1 (New England Biolabs) carrier DNA adds 5ul 10X damping fluid, 1ul SapI restriction endonuclease and 1ul PstI restriction endonuclease (New England Biolab), add deionized water again to final volume 48ul, 37 ℃ are incubated 1 hour, and then add 1ul SapI restriction endonuclease and 1ul PstI restriction endonuclease, 37 ℃ are incubated 1 hour again, and then add 37 ℃ of insulations of 1ul CIP (Calf IntestinalPhosphatase, New England Biolab company) 20 minutes.PCR product after enzyme is cut separates through 1% agar electrophoresis with carrier, cut out the 150bp size and contain the DNA band of A β and the carrier band of 6.6kb, goal gene and carrier purifying after the AGROSE GEL EXTRACTIONKIT that re-uses QIAGEN cuts enzyme: weigh up the agar weight that contains target DNA, add 3 times of QG damping fluids that the volume test kit provides,, add 1 times again and behind the Virahol of agar volume, cross the MiniElute affinity column so that agar dissolves 50 ℃ of water bath heat preservations 10 minutes.Use the Eppendorff whizzer after centrifugal one minute, target DNA can be trapped on the affinity column.The QG damping fluid that uses the 500ul test kit to provide, then the PE solution centrifugal that provides of 750ul test kit clean the MiniElute affinity column each once, use 20ul 10mMpH8.5 Tris-Cl buffer solution elution target DNA then.After obtaining restriction endonuclease is handled and purifying is crossed fusion antibody dna and carrier, use the RAPID DNA LIGATIONKIT of ROCHE company that they are linked together: the fusion antibody dna that the above-mentioned purifying of 3ul is crossed adds the 1ul carrier, add the 5X DNA mixing solutions that the 1ul test kit provides again, 23-25 ℃ of room temperature insulation of ligase enzyme (ligase) that the 2X that adds the 5ul test kit behind two kinds of DNA again and provide connects damping fluid and 0.5ul test kit and provide is provided got final product in 5-10 minute.Use the above-mentioned DNA of connecting of 2ul to add 20-40ul competence DH5 α bacterial cell, 4 ℃ are incubated 20 minutes on ice, 42 ℃ of water-bath heat-shockeds 1 minute, 4 ℃ were cooled off 2 minutes, add SOC nutrient solution (Invitrogen company) 200ul, 37 ℃ of shaking tables were cultivated 45 minutes, were coated with the LB agar plate that contains penbritin then.Select positive bacterium colony to increase in a small amount and extract DNA (the QIASPINPLASMID MINI KIT of QIAGEN company), cut through SapI and PstI enzyme, after the exactness of plasmid construction is expressed in dna sequence analysis checking, carry out DNA increase in a large number (the QIAFILTER PLASMID MAXI KIT of QIAGENQ company).Fig. 2 a is the synoptic diagram with the carrier that contains fusion gene of this method structure.

Two, escherichia coli expression fusion rotein and A β separation and purification

With the plasmid expression vector Twin1-N-A β 40 that clones target gene that contains that builds, transform in E.coli BL21 (DE3) system and carry out abduction delivering and purifying, the concrete operations step is as follows:

(1) abduction delivering of fusion rotein

Cloning vector is transformed into E.coli BL21 (DE3) (Novagen company) with Calcium Chloride Method, in 1.5% agar plate that contains penbritin (100ug/ml), 37 ℃ of incubated overnight.(Tryptone 0.2%, yeast extract 1.5%, Na at the TP that contains penbritin (100ug/ml) to choose mono-clonal next day ₂HPO ₄0.2%, KH ₂PO ₄0.1%, NaCl 0.8%, and with preceding adding 20% glucose, making final concentration is 0.2%) in the substratum, 37 ℃ of shaking culture are spent the night.Overnight culture is inoculated in the 1L fresh culture by 1/100, and 37 ℃ are continued to cultivate, treat OD ₆₀₀Reach and carry out abduction delivering between the 0.5-0.7, inductor IPTG concentration is 0.8mM, induces 5 hours for 37 ℃.Centrifugal then 6000rpm, 15 minutes results thalline can be put-20 ℃ of preservations.

(2) washing of inclusion body, dissolving, renaturation

Thalline is resuspended among the Buffer A (50mM Tris pH8.5,50mM NaCl, 5mMEDTA, 0.1mM PMSF) (10ml/g), and ultrasonic wave is broken bacterium (400W worked 30 seconds, interval 60 seconds, 20 minutes).Centrifugal 15000rpm, 20 minutes, discard supernatant, with precipitation be resuspended in Buffer B (50mM Tris pH8.5,50mM NaCl, 5mMEDTA, 0.3%SDS, 5%Tritonx-100, Glycerin10%) in (10ml/g), put room temperature spent the night in 3 hours or 4 ℃.Wash once with Buffer B then, Buffer A washes twice.Inclusion body after washing BufferC (4M urea, 20mM Tris pH8.5,50mM NaCl, 1mM EDTA) dissolving.Inclusion body successive after the dissolving uses the BufferC that contains 2M, 1M, 0.5M, 0M urea in 4 ℃ of dialysis renaturation (8～12h/ time).Centrifugal 12000rpm collected supernatant in 15 minutes, put 4 ℃ and prepared chromatographic separation.Extract that small amount of sample adds that the SDS sample-loading buffer carries out that SDS-PAGE separates and staining analysis (Fig. 3 a).

(3) separation, the purifying of purpose A β 40 polypeptide

With 10 column volume Buffer B ₁(20mM Tris PH8.5,50mM NaCl, 1mM EDTA) balance Chitin Bead chromatography column.Slowly go up sample (flow velocity is no more than 0.5-1.0ml/min), 15 column volume Buffer B then ₁The flushing chromatography column, (flow velocity 2ml/min).Use 3 column volume Buffer B ₂(20mM Tris PH7.0,50mM NaCl, 1mM EDTA) is fast by replacing Buffer B ₁, spend the night in room temperature (25 ℃) cracking.Next day, use Buffer B ₂Elution samples is pressed the 2ml/ pipe and is collected.The BufferB that contains 1%SDS then with 3 column volumes ₁Room temperature is cleaned chromatography column.

(4) lyophilize of sample

It is 2000 dialysis tubing that the sample of collecting is placed the size-exclusion amount, is concentrated into 0.5mg/ml with PEG8000 absorption.Concentrate back sample NH ₄AC (20mM, pH7.5), 4 ℃ of dialysis ,-80 ℃ are freezing.Carry out vacuum lyophilization.SDS-PAGE electrophoresis initial analysis molecular weight and purity (Fig. 3 b).

Embodiment 2:A β 40 carboxyl terminales merge the bacterial expression of the fusion rotein of Intein 1

One, makes up the DNA of coding A β-Intein1 fusion rotein

Use PCR method to become precursor protein (APP) selective amplification A β 40 polypeptide genes from human amyloid.Select the EXPEND HIGH FIDELITY PCR KIT of ROCHE company for use.The PCR reaction comprises 38ul deionized water, the 10X damping fluid that the 5ul test kit provides, 3ulDMSO, 1ul dNTP (10mM dATP, 10mM dCTP, 10mM dGTP, 10mMdTTP), the hybrid dna polysaccharase that the 1ul test kit provides, 1ul 10ng/ml contain the DNA plasmid of people APP as template.1ul 10pM forward primer and 1ul 10pM inverse pairs primer.Select the forward primer Ab-C-Nde-F that contains the Nde1 restriction enzyme site for use:

GGAATTC CATATGGATGCAGAATTCCGA；

Select the reverse primer A β 40-C-Sap-R that contains the Sap1 restriction enzyme site for use:

GTGCATT GCTCTTCTGCATTAGACAACACCGCCCACCATG (A β 40 polypeptide)

Perhaps A β 42-C-Sap-R:

CCAGTCTA GCTCTTCTGCACTACGCTATGACAACACCGCC (A β 42 polypeptide).

The primer combinations of pairs of PCR reaction is summarized as follows:

Aβ40：Aβ-C-Nde-F+Aβ40-C-Sap-R

Aβ42：Aβ-C-Nde-F+Aβ42-C-Sap-R

The PCR reaction is used 94 ℃, 30 seconds; 54 ℃, 30 seconds; 72 ℃, 1 minute; Totally 30 loop cycles.Extract 2ul PCR product and add 3ul deionized water and 1ul electrophoresis solution (SigmaG2526), after the separation of 1% agar electrophoresis confirms that the PCR product contains the DNA of target gene size, all the other PCR products are used the QIAQUIK PCRPURIFICATION KIT purifying of QIAGEN company: a PCR reaction product is mixed with the PB damping fluid that five parts of test kits provide, and the small-sized centrifugal post that test kit provided of packing into then is centrifugal; Re-use that 0.5ml PB damping fluid is centrifugal washes post once; The EB damping fluid that is provided with the 50ul test kit is adorned the centrifugal wash-out of post again.Through the DNA concentration determination, draw the PCR product of 1ug purifying, add 2ul 10X damping fluid, 1ul SapI restriction endonuclease and 1ul NdeI restriction endonuclease (New EnglandBiolabs) add deionized water again to final volume 20ul, and 37 ℃ are incubated 2 hours.1ugTwin1 (New England Biolabs) carrier DNA adds 5ul 10X damping fluid, 1ul SapI restriction endonuclease and 1ul NdeI restriction endonuclease (New England Biolab), add deionized water again to final volume 48ul, 37 ℃ are incubated 1 hour, and then add 1ul SapI restriction endonuclease and 1ul NdeI restriction endonuclease, 37 ℃ are incubated 1 hour again, and then add 37 ℃ of insulations of 1ul CIP (Calf IntestinalPhosphatase, New England Biolab company) 20 minutes.PCR product after enzyme is cut separates through 1% agar electrophoresis with carrier, cut out the 150bp size and contain the DNA band of A β and the carrier band of 6.6kb, goal gene and carrier purifying after the AGROSE GEL EXTRACTIONKIT that re-uses QIAGEN cuts enzyme: weigh up the agar weight that contains target DNA, the QG damping fluid that the test kit of 3 times of volumes of adding provides,, add 1 times again and behind the Virahol of agar volume, cross the MiniElute affinity column so that agar dissolves 50 ℃ of water bath heat preservations 10 minutes.Use the Eppendorff whizzer after centrifugal one minute, target DNA can be trapped on the affinity column.The QG damping fluid that uses the 500ul test kit to provide, then the PE solution centrifugal that provides of 750ul test kit clean the MiniElute affinity column each once, use 20ul 10mMpH8.5 Tris-Cl buffer solution elution target DNA then.After obtaining restriction endonuclease is handled and purifying is crossed fusion antibody dna and carrier, use the RAPID DNA LIGATIONKIT of ROCHE company that they are linked together: the fusion antibody dna that the above-mentioned purifying of 3ul is crossed adds the 1ul carrier, add the 5X DNA mixing solutions that the 1ul test kit provides again, the 2X that adds the 5ul test kit behind two kinds of DNA again and provide is provided connects the ligase enzyme (ligase) that damping fluid and 0.5ul test kit provide, mix the back and getting final product in 5-10 minute 23-25 ℃ of room temperature insulation.Use the above-mentioned DNA of connecting of 2ul to add 20-40ul competence bacillus coli DH 5 alpha bacterial cell, 4 ℃ are incubated 20 minutes on ice, 42 ℃ of water-bath heat-shockeds 1 minute, 4 ℃ were cooled off 2 minutes, add SOC nutrient solution (Invitrogen company) 200ul, 37 ℃ of shaking tables were cultivated 45 minutes, were coated with the LB agar plate that contains penbritin then.Select positive bacterium colony to increase in a small amount and extract DNA (the QIASPIN PLASMID MINI KIT of QIAGEN company), cut through SapI and NdeI enzyme, after the exactness of plasmid construction is expressed in dna sequence analysis checking, carry out DNA increase in a large number (the QIAFILTER PLASMID MAXI KIT of QIAGENQ company).Fig. 2 b is the synoptic diagram with the carrier that contains fusion gene of this method structure.

Two, escherichia coli expression fusion rotein and A β separation and purification

Transform into and carry out abduction delivering and purifying in E.coli BL21 (DE3) system with the plasmid expression vector A β 40-C-Twin1 that clones target gene that contains that builds.The concrete operations step is as follows:

The abduction delivering of fusion rotein, the washing of inclusion body, dissolving, renaturation, all identical with the bacterial expression of above-mentioned A β aminoterminal fusion Intein.

The separation of purpose A beta polypeptides, purifying:

With 10 column volume Buffer B ₁(20mM Tris PH8.5,50mM NaCl, 1mMEDTA) balance Chitin Bead chromatography column.Slowly go up sample (flow velocity is no more than 0.5-1.0ml/min), 15 column volume Buffer B then ₁The flushing chromatography column, (flow velocity 2ml/min).

Use 3 column volume Buffer B ₃(20mM Tris PH8.5,500mM NaCl, 1mM EDTA, 40mM DTT) is fast by replacing Buffer B ₁, spend the night in 4 ℃ of cracking.Next day, use Buffer B ₃Elution samples is pressed the 2ml/ pipe and is collected.The BufferB that contains 1%SDS then with 3 column volumes ₁Room temperature is cleaned chromatography column.

The lyophilize program of sample is also identical with the proteic cracking purifying of above-mentioned A β aminoterminal fusion Intein.

Reference:

Bard F etc., Nature Medicine 6:916-919.

Janus C etc., Nature 408:979-982;

Lemere C etc., DNA Cell Biol.20:705-711

Levy E etc., Science248:1124-1126.

Morgan D etc., Nature408:915-916.

Poduslo JF etc., Neuroreport 12:3197-3200

Schenk D etc., Nature400:116-117.

Weiner H etc., Annals Neurology 48:567-579

Claims (14)

1. the method for a production senile dementia morbid substance amyloidosis polypeptide β (A β), this method comprises:

(1) with described A beta polypeptides coding region and easy expressed proteins gene fusion in bacterium;

(2) fusion gene is placed bacterial expression vector;

(3) transfection host bacterium is expressed fusion product;

(4) purified fusion protein separates the A beta polypeptides from fusion rotein.

2. method according to claim 1, wherein the described fusion of step (1) is meant that easy expressed proteins gene fusion is to the aminoterminal or the carboxyl terminal of A beta polypeptides in bacterium.

3. method according to claim 1 and 2, wherein the described bacterial expression vector of step (2) contains the expression promotor of bacterium.

4. method according to claim 3, wherein said bacterial expression vector is Twin1.

5. according to each described method of claim 1-4, wherein host bacterium is intestinal bacteria.

6. method according to claim 5, wherein host bacterium is e. coli bl21 (DE3).

7. according to each described method of claim 1-6, wherein the A beta polypeptides is a people A beta polypeptides sequence deutero-.

8. method according to claim 7, wherein the A beta polypeptides has following amino acid sequences:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(Aβ42)

Or DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (A β 40)

Or have continuous 5 amino acid whose sequences in above-mentioned arbitrary aminoacid sequence.

9. according to each described method of claim 1-8, wherein said fusion rotein has the polypeptide that will merge with it under certain condition to cut off or the function of enzymolysis at position of fusion.

10. method according to claim 9, wherein said fusion rotein is one of following albumen: Intein, GST, Lac Z, or other has the bacterioprotein or the synthetic protein in enzymolysis or hydrolysis site at albumen and polypeptide corresponding circle of sensation.

11. method according to claim 10, wherein said Intein fusion rotein has following protein sequence:

MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALW QLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEIWAINEQTMKL ESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDELSLKEHIALP RKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGPHNFVANDIIV HN or/and

CITGDALVALPEGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTVEGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFARGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVTDAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ

12. an A beta fusion proteins has following aminoacid sequence:

MKIEEGKLTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSN

VPALWQLQNNGNNGLELRESGAISGDSLISLASTGKRVSIKDLLDEKDFEIWAI

NEQTMKLESAKVSRVFCTGKKLVYILKTRLGRTIKATANHRFLTIDGWKRLDE

LSLKEHIALPRKLESSSLQLSPEIEKLSQSDIYWDSIVSITETGVEEVFDLTVPGP

HNFVANDIIVHN DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

Or

MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVCITGDALVALP

EGESVRIADIVPGARPNSDNAIDLKVLDRHGNPVLADRLFHSGEHPVYTVRTV

EGLRVTGTANHPLLCLVDVAGVPTLLWKLIDEIKPGDYAVIQRSAFSVDCAGFA

RGKPEFAPTTYTVGVPGLVRFLEAHHRDPDAQAIADELTDGRFYYAKVASVT

DAGVQPVYSLRVDTADHAFITNGFVSHATGLTGLNSGLTTNPGVSAWQVNTA

YTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ

13. an A beta polypeptides has following amino acid sequences:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA(Aβ42)

Or DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (A β 40)

Or have continuous 5 amino acid whose sequences in above-mentioned arbitrary aminoacid sequence.

14. the described A beta polypeptides of claim 13 is used for the treatment of and prevents application in the medicine of senile dementia in preparation.

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