CN101538329B - Variable region sequence for novel anti-beta amyloid polypeptide antibody and coding sequence thereof - Google Patents

Abstract

The invention relates to a variable region sequence for a novel anti-beta amyloid polypeptide antibody and a coding sequence thereof. The invention utilizes phage display technology to perform multiple screening and enrichment on a human-originated phage single-chain antibody library to obtain phages with Abeta specificity, prepares a phage antibody by culturing the phages, and identifies the acquisition of positive clone. Then an anti-Abeta scFv segment which obtains the solubility is expressed in Escherichia coli, and a corresponding coding sequence of the anti-Abeta scFv segment is obtained through the sequence measurement. Therefore, the invention provides the novel anti-Abeta scFv segment and the coding sequence thereof.

Description

Variable region sequences and the encoding sequence thereof of new directed against amyloid-beta polypeptide antibody

Technical field

The present invention relates to the antibody of directed against amyloid-beta polypeptide, further relate to the specificity scFv antibody of new directed against amyloid-beta polypeptide, and the aminoacid sequence of variable region and dna encoding sequence.

Background technology

Alzheimer's disease (Alzheimer disease, AD) be a kind of in the elderly carrying out property dysmnesia and hypophrenia be the central nervous system degenerative disease of main clinical characteristics.Senile plaque, neurofibrillary tangle and neuron loss are the three large main pathological characters of AD.Wherein the main component of senile plaque is beta-amyloid polypeptide 1-(beta-amyloid peptide, A β) assembles the fiber that forms, and A β is considered to cause main morbid substance [the Frost D.Co-incorporation of A beta 40and A beta42 to formmixed pre-fibrillar aggregates.Eur J Biochem of neuronal damage and cognitive function decline, 2003,270 (4): 654-663].A β mainly comprises A β 40 and 42 two kinds of molecules of A β, is comprised of 39～43 amino acid.It is generally acknowledged that A β is because the gene of coding APP is undergone mutation or other reasons when causing the beta-secretase activity unusually to increase, and cuts generation jointly with gamma-secretase.Also there is now viewpoint to think that the A β that is higher than normal people's concentration in AD patient's brain may be because the transhipment of A β is removed occurs due to the obstacle.Studies confirm that increasing of A β content descends closely related with cognitive ability in brain and the cerebrospinal fluid.Hydrophobic region in the A beta structure plays an important role for the formation of regulating the A beta, and its 1～28 amino acids is hydrophilic N-terminal, is hydrophobic C-terminal after 29.Because hydrophobic amino acid mainly is positioned at carboxyl terminal, so the length of carboxyl terminal determines its deliquescent height, and impact is gathered into the speed of fiber.The fiber that A β assembles formation mutually has toxicity, can inspire cascade of response of inflammation, axonal injury, cynapse is lost and the pathological changes such as apoptosis, to cause neuronal degeneration and even form dull-witted important factor [McGeer EG, McGeer PL, Inflammatory processes in Alzheimer ' s disease[J], ProgNeuropsychopharmacol Biol Psychiatry, 2003,27 (5) 1741-749].

Along with improving constantly of whole world aging population degree, the sickness rate of this disease is given society and family at material and has all been brought mentally tremendous influence also in gradually rising.How finding a kind of medicine that can treat this disease is a urgent need.But there is no so far the treatment measure of special efficacy, clinically mainly take symptomatic treatment and rehabilitation therapy as main, as using acetylcholinesterase depressant, descend to remedy the vagusstoff neurohumor that brings because of neurodegeneration.But these therapies can not fundamentally be treated AD.And for the keying action of A beta polypeptides in morbidity, research and develop the actual application value that anti-A beta polypeptides antibody just has treatment AD.

Anti-amyloid beta antibodies treatment AD, mainly by two kinds of approach remove A β in the body [Sheng Shuli, chief editor. senile dementia and relative disease. Beijing: scientific and technical literature press, 2006:179-200].The one, antibody enters the interior A beta in senile plaque of brain through hemato encephalic barrier and is combined, and microglia is combined with the Fc of antibody fragment by the Fc acceptor, engulfs, removes A beta wherein; The 2nd, antibody is combined with the A of solubility β in peripheral circulation, increases the concentration difference of the inside and outside A β of hemato encephalic barrier, promotes the A β outflow in the brain, thereby reduces the A β level in the brain, reduces gathering and the deposition of A β in brain.At present, proved that anti-amyloid beta antibodies (comprising many anti-, monoclonal antibody, antibody fragment etc.) has sure effect to removing A β in vitro tests and animal body, the anti-amyloid beta antibodies treatment has potential using value.

The in vitro tests progress mainly is the effect of observing the anti-amyloid beta antibodies neutralization or removing A β.The Legleiter philosophy is hatched with 1: 10 mol ratio with A β 1-42 with m266 and 3D6 monoclonal antibody, observing A beta formational situation with atomic force microscope (AFM) after several days finds: the formation that two kinds of monoclonal antibodies can both establishment A beta, some [Legleiter J of m266 better effects if wherein, Czilli DL, Gitter B, et al.Effect of different anti-Abeta antibodieson Abeta fibrillogenesis as assessed by atomic force microscopy.J MolBiol, 2004,335 (4): 997-1006].Anti-A β antibody can improve cell to the tolerance of A β, PC12 cell and the hippocampal neuron of cultivating had provide protection [Yang Zhiyong; Wang overseas Chinese; Xu Jie etc. the characteristic of anti-amyloid in the alzheimer's disease human peripheral. anatomical research; 2005; 27:83-87.Du Y; Wei X; Dodel R; et al.Human anti-β-amyloidantibodies block β-amyloid fibril formation and prevent β-amyloid-induced neurotoxicity.Brain; 2003,126 (9): 1935-9].In order to detect the effect of cleaning antibody patch, Bard etc. with anti-A β monoclonal antibody (10D5,3D6) through abdominal injection to the PDAPP transgenic mice, put to death mouse after six months, its freezing brain section was hatched 24 hours with microglia, found that by immunostaining the external source microglia can engulf the A beta in the patch; And control group, what patch kept complete and had no microglia engulfs phenomenon [BardF, Cannon C, Barbour R, et al.Peripherally administered antibodies againstamyloid beta-peptide enter the central nervous system and reducepathology in a mouse model of Alzheimer disease.NatMed, 2000,6 (8): 916-9].Intravenous immunoglobulin (the IVIG that extracts in the bibliographical information healthy human body is arranged, intravenous immunoglobulin) contains anti-amyloid beta antibodies [Dodel RC in, DuY, Depboylu C, et al.Intravenous immunoglobulins containing antibodiesagainst β-amyloid for the treatment of Alzheimer ' s disease.Journal ofNeurology Neurosurgery and Psychiatry, 2004,75:1472-1474], these antibody can not only suppress the A beta peptide aggregation and become fiber, can also make the A beta degraded that has formed, can also improve simultaneously microglia to the ability of amyloid plaques migration, and the mediation microglia engulfs, degraded A β [Solomon B.Intravenous immunoglobulin and Alzheimer ' s diseaseimmunotherapy.Curr Opin Mol Ther, 2007,9 (1): 79-85.Istrin G, BosisE, Solomon B, et al.Intravenous immunoglobulin enhances the clearance offibrillar amyloid-beta peptide.J Neurosci Res, 2006,84 (2): 434-43].The propositions such as Fukuchi, the scFv fragment of anti-amyloid beta antibodies also can suppress A beta oligomers (A β oligomers) to toxic action [the Fukuchi K of HEK293 cell, Accavitti-Loper MA, Kim HD, etal.Amelioration of amyloid load by anti-Abeta single-chain antibody inAlzheimer mouse model.Biochem Biophys Res Commun, 2006,344 (1): 79-86].Above-mentioned in vitro tests shows, anti-amyloid beta antibodies can by the receptor-mediated microglia of the Fc A beta of engulfing, degrade, also can suppress the A beta peptide aggregation and become fiber or patch by other means.

Animal experiment is the prerequisite that antibody enters clinical study, and the most laboratory animal is adopted the APP transgenic mouse.Then antibody observe antibody distribution, the position that plays a role and action effect in vivo through periphery (such as vein, abdominal cavity) or in cranial cavity imports animal body.The discovery anti-amyloid beta antibodies such as Schenk can be removed formation [the Schenk D that the interior senile plaque of mouse brain also can prevent senile plaque in 1999, Barbour R, Dunn W, et al.Immunization with amyloid--betaattenuates Alzheimer-disease-like pathology in the PDAPP mouse.Nature, 1999,400 (8): 173-7].DeMattos etc. will resist A β monoclonal antibody m266 to inject through peripheral vein only can be in cerebral tissue be expressed the PDAPP transgenic mice body of APP, the result shows in the blood plasma 1000 times of A β Enrichments, A β accumulates obvious minimizing in the cerebral tissue, illustrate in the antibody capable and peripheral circulation in A β, make the A β in the brain progressively diffuse into blood and be eliminated [DeMattosRB, Bales KR, Cummins DJ, et al.Peripheral anti-A beta antibody alters CNSand plasma A beta clearance and decreases brain A beta burden in a mousemodel of Alzheimer ' s disease.Proc Natl Acad Sci U S A, 2001,98 (15): 8850-5].Bard etc. with anti-amyloid beta antibodies through abdominal injection in the PDAPP transgenic mice body, iconography finds that patch reduces more than 80% in the brain after six months, antibody capable in conjunction with amyloid plaques, and activates microglia by hemato encephalic barrier, has promoted the removing of previous already present amyloid plaques.Chauhan etc. directly are expelled to antibody in the third ventricle, and different time sections checks the cerebral tissue of mouse after injection, detected result shows, during to 4th week, the density ratio control group mice for the treatment of group amyloid has reduced 67%, the 4th week effect peaks after simultaneously also having proved the intracerebral ventricle injection of anti-A β monoclonal antibody, begin [the ChauhanNB that disappears after eight weeks, Siegel GJ.Intracerebroventricular passive immunization in transgenicmouse models of Alzheimer ' s disease.Expert Rev Vaccines, 2004,3 (6): 717-25.Chauhan NB.Intracerebroventricular passive immunizationwith anti-oligoAbeta antibody in TgCRND8.J Neurosci Res, 2007,85 (2): 451-63].Recently find that the A beta oligomers also has stronger toxicity in the brain; Ma etc. are expelled to anti-amyloid beta antibodies in the Tg2576 Transgenic Mice Brain; find that it can remove A beta oligomers in the mouse brain; the Cranial nerve injury as birth trauma that watches for animals there is positive effect [Ma QL; Lim GP; Harris-WhiteME; et al.Antibodies against beta-amyloid reduce Abeta oligomers; glycogensynthase kinase-3beta activation and tau phosphorylation in vivo and invitro; J Neurosci Res; 2006,83 (3): 374-84].The discoveries such as Levites, after the monoclonal antibody of anti-A β 1-40 and A β 1-42 is injected in the APP transgenic mice body, the amyloid plaques total amount reduces by about 50% [Levites Y in the brain, Smithson LA, Price RW, et al.Insights into themechanisms of action of anti-Abeta antibodies in Alzheimer ' s diseasemouse models.FASEB J, 2006,20 (14): 2576-8.Levites Y, Das P, PriceRW, et al.Anti-Abeta42-and anti-Abeta40-specific mAbs attenuate amyloiddeposition in an Alzheimer disease mouse model.J Clin Invest, 2006,116 (1): 193-201].Except above-mentioned whole antibody, antibody fragment also can be brought into play effect in animal body.The researchist is the anti-A β of successful Application monoclonal antibody fragment, such as special F (ab ') 2 fragments, scFv fragment etc., through abdominal injection, intracranial injection or in the different way of administration such as adenovirus mediated imports APP transgenic mice body, all obviously reduced old patch [the Tamura Y in the brain, Hamajima K, Matsui K, et al.The F (ab) ' 2 fragment of anAbeta-specific monoclonal antibody reduces Abeta deposits in thebrain.Neurobiol Dis, 2005,20 (2): 541-9.Fukuchi K, Tahara K, KimHD, et al.Anti-Abeta single-chain antibody delivery via adeno-associatedvirus for treatment of Alzheimer ' s disease.Neurobiol Dis, 2006,23 (3): 502-11].

Anti-amyloid beta antibodies treatment AD is successfully in vitro tests and animal experiment.But the antibody that finally will be used for human body therapy must be through humanized, causes the limitation of human antimouse antibody (HAMA) reaction at human body to overcome mouse monoclonal antibody.Obtaining humanized antibody generally is to adopt to carry out mouse source antibody humanization modified, but this method and technology component difficulty is high, even also be difficult to obtain hundred-percent humanization after transforming, and might in transformation process, make the avidity of tiring of original antibody reduce [Mike Clark.Antibody humanization:a caseofthe ' Emperor ' s new clothes? Immunol Today, 2000,21 (8): 397-402].Other method is utilized directly screening [Hennie RH.Selecting and screening recombinant antibody bibraries.Nature Biotechnol from the human antibody gene pool of phage display technique exactly, 2005,23 (9): 1105-1116], gene in the natural antibody storehouse comes from a complete set of variable region gene of human B cell (peripheral blood lymphocyte or splenocyte) without immunity, have enough capacity and diversity widely, usually contain 10 ⁶-10 ⁹The antibody molecule clone of individual different antigen-binding specificities, can select 1,000,000 and even hundreds of millions molecules, and each takes turns " adsorbing a wash-out one amplification " can make the concentrated 50-1000 of purpose antibody concentration doubly, make the stronger clone of antigen-specific obtain enrichment, be very easy to from antibody library, screen specific antibody.And the antibody that therefrom screens has been complete people source, need not to carry out humanization modified again.Single-chain antibody (scFv) is the small molecular antibody fragment of utilizing the phage display technique preparation, it does not have the Fc section of complete antibody, only comprise VH and be connected middle connection skin (Linker) with the VL fragment, the antigen affinity that has but kept preferably complete antibody is the minimum antibody fragment with complete antigen binding site.

ScFv compares with complete antibody, has following advantage: (1) scFv molecule is little, immunogenicity is low.Its relative molecular weight only has the sixth of complete antibody, is applied to human body and is difficult for producing the allos reaction.(2) the scFv penetration power is strong.After entering body, can pass rapidly vessel wall and organize barrier to arrive target cell and target tissue, be conducive to disease treatment.(3) scFv can express in prokaryotic system, is easy to genetic manipulation and genetically engineered is produced in a large number, and is with low cost; Also available gene engineering method makes up the various fusion roteins that are connected with other effector molecules such as medicine, cytokine etc.(4) scFv does not have the Fc section, does not need the Fc receptors bind with non-target cell, is conducive to the carrier as targeted drug.(5) scFv transformation period weak point is easily removed from blood, and the kidney accumulation is few, in view of the above advantage that scFv possesses, makes it obtain using comparatively widely.At present, the research of scFv and utilize genetically engineered production and use progress swift and violent has occupied the share more than 30% in the clinical trial of biological preparation medicine.But scFv transformation period weak point also is its shortcoming, and another shortcoming of scFv is that less stable is easy to degraded.

Whole antibody is the constant region FC fragment gene of the upper people's antibody of variable region gene connection to be recombinated form, and whole antibody is because have constant region and FC section, and molecular weight is larger, stability is higher and the transformation period is longer; Because whole antibody is the bivalent conjugated antigen, and scFv is the unit price conjugated antigen, so avidity can be larger than scFv in theory; Moreover the constant region of whole antibody and FC section can corresponding activating complements, are conducive to directly remove antigen, or produce the immune clearance effect with the Cell binding with FC acceptor, so have more in theory using value.But whole antibody can increase the difficulty of passing hemato encephalic barrier, but have and studies show that truly having partial antibody can pass hemato encephalic barrier plays a role, even and the antibody of staying in the circulation of blood also can be combined and removing by the A β in blood, A beta monomers after A β in the blood reduces in the brain can constantly be penetrated in the blood vessel, thereby the A beta substance in the brain is reduced, play equally effect [the DeMattos RB that reduces A β pathology material in the brain, Bales KR, Cummins DJ, et al.Peripheral anti-A β antibody alters CNS and plasma A β clearance and decreases brain A β burden in a mouse model of Alzheimer ' s disease.Proc Natl Acad Sci USA.2001,98 (15): 8850-8855].

Summary of the invention

In view of the situation of present treatment AD, find new anti-amyloid beta antibodies help lend some impetus to and improve to the treatment AD treatment.The invention provides new humanization anti-amyloid beta antibodies variable region fragment and encoding sequence thereof.

The invention provides a kind of human source anti-A β specificity scFv antibody that comprises variable region of heavy chain, variable region of light chain and catenation sequence, the aminoacid sequence of its variable region of heavy chain is shown in SEQ ID NO:2, and the aminoacid sequence of variable region of light chain is shown in SEQ ID NO:4.

The present invention also provides a kind of DNA of the described human source anti-A β specificity scFv antibody of encoding, and its nucleotide sequence is comprised of the DNA of the DNA shown in the DNA shown in the SEQ ID NO:1, the SEQ ID NO:3 and coding catenation sequence.

On the other hand, the present invention also provides the DNA of the weight chain variabl area sequence of the anti-A β specificity scFv antibody of encoding, and its nucleotide sequence is shown in SEQ ID NO:1; The encode DNA of light chain variable region sequence of anti-A β specificity scFv antibody, its nucleotide sequence is shown in SEQ IDNO:3.

Again on the one hand, the invention provides recombinant vectors and the corresponding recombinant host cell thereof that comprises described DNA.Recombinant host cell is intestinal bacteria (Escherichia coli) preferably.

Further, be the invention provides on March 12nd, 2008 and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is the recombination bacillus coli of CGMCC No.2399.

The beneficial effect that the application provides has been to provide a kind of new anti-amyloid beta antibodies, and the anti-amyloid beta antibodies that obtains is scFv.

Description of drawings

Fig. 1: the ELISA detected result of positive phage clones.

Fig. 2: positive phagemid PCR result, wherein M is marker; 1-7 is ELISA 7 the higher phage clones of tiring.

Fig. 3: positive colony solubility expression whole bacterial protein electrophoresis result, wherein M is marker; 2,4,6,8,9,11,13 is that A10, D8, F2, B9, E6, F4, the G2 that induces bites Jun body clone; 1,3,5,7,10,12,14 for biting accordingly Jun body clone but induce without IPTG.

The distribution of scFv antibody in each component of Fig. 4: A10 clone solubility expression, wherein 1 is the culture supernatant of not inducing; 2 culture supernatant of inducing for IPTG; 3 is born of the same parents' pericentral siphon of not inducing; 4 born of the same parents' pericentral siphons of inducing for IPTG; 5 is the whole bacterial protein of not inducing; 6 whole bacterial proteins of inducing for IPTG.

Fig. 5: induce strain and do not induce the ELISA result of soluble scFv in each component of strain.

Fig. 6: the gene variable region sequences of anti-amyloid beta antibodies antibody and corresponding protein sequence.

Those skilled in the art can understand, according to the disclosed content of the application in conjunction with art technology general knowledge, the disclosed dna sequence dna of the application and aminoacid sequence can be synthetic according to this area additive method commonly used, and for example the method by chemosynthesis obtains the disclosed sequence of the application.And those skilled in the art can be building up to the new dna sequence dna that the application obtains in any suitable carrier, the host cell.Following content only is the exemplary illustration to the claimed scope of the application, and those skilled in the art can make multiple change and modification to the application's invention according to disclosed content, and it also should belong among the claimed scope of the application.

Embodiment

Embodiment 1: the screening of mono-clonal phage antibody:

Utilize display technique of bacteriophage that people source phage antibody library is carried out many wheel enrichment screenings, carried out the enrichment screening that four-wheel " adsorbs a wash-out one amplification " take the alzheimer's disease A β of purifying as antigen, show that every screening rate of recovery of taking turns is and increases progressively trend, illustrate through after the screening, have the specific phage of A β and obtained highly enriched (table 1).Method is as follows:

(1) amplification of phage antibody library: get the former storehouse of 100 μ l bacterium liquid, (contain 100 μ g/ml penbritins, in 2 * YT) substratum of 1% glucose, 37 ℃ of shaking culture are to logarithmic phase to be inoculated into 100ml 2 * YT-AG.Add 10 ¹²Pfu (plaque-forming unit) helper phage M13KO7 is in nutrient solution, and 37 ℃ of water-bath 30min abandon supernatant after centrifugal for 4 ℃.(contain 100 μ g/ml penbritins, 2 * YT) resuspended precipitations of 50 μ g/ml kantlex, 37 ℃ of overnight incubation with 100ml 2 * YT-AK.4 ℃ centrifugal, and supernatant is the phage antibody library after the amplification.Add 100mlPEG/NaCl in supernatant, the centrifugal supernatant of abandoning behind the ice bath 1h is with the resuspended precipitation of PBS, 4 ℃ of preservations.

(2) enrichment of antibody library screening: with 8 holes of A β 1-42 (400ng/ hole) coated elisa plate of purifying, 4 ℃ of placements are spent the night.37 ℃ of sealings of inferior daily 2%MPBS (PBS that contains 2% skim-milk) 2h abandons supernatant, and after the hole flushing, every hole adds the phage antibody supernatant after the 100 μ l amplification, hatches 2h for 37 ℃.Abandon supernatant, (first round washes 3 times when screening in hole flushing, second takes turns and washes 5 times, third round is washed later on more than 10 times), every hole adds 100 μ l 100mmol/L glycine-hydrochloric acid (μ H2.2) wash-out specific phage antibody, behind the incubated at room 10min, add immediately the special phage antibody that elutes of 1mol/LTris (pH7.4) neutralization.Elutriant is added in the logarithmic phase TG1 bacterium liquid of the fresh preparation of 2ml, behind 37 ℃ of water-bath 30min, the bacterium liquid that takes a morsel is surveyed the phage antibody titre, more than all be coated with the TYE-AG flat board, 37 ℃ of overnight incubation, scrape bacterium colony from the TYE plate next day, and resulting bacterium colony is amplified and uses phage for the preparation of the next round screening.Carry out altogether the four-wheel screening.Calculating every the wheel drops into and quantum of output.

Table 1 is respectively taken turns the rate of recovery of enrichment screening pnagus medius antibody

Annotate: the rate of recovery (the %)=output phagocytosis scale of construction/input phagocytosis scale of construction * 100%

Embodiment 2: the mono-clonal phage antibody is detected:

80 single bacterias clones of picking at random on the TYE-AG flat board of incubated overnight cultivate the preparation phage antibody after the fourth round screening.Detect the ELISA method of using: antigen A β coated elisa plate (0.4 μ g/ hole), seal backward every hole and add 100 μ l mono-clonal phage antibody supernatant liquors, 37 ℃ of incubation 2h.The HRP mark mouse-anti M13 antibody that adds 1: 5000 after the washing, 37 ℃ of incubation 1h.Positive control is primary antibodie (mouse-anti people A β), two anti-(sheep anti mouse lgG); Negative control only adds 2%MPBS.Take OPD as substrate, 2M sulfuric acid termination reaction detects through microplate reader OD490nm.With greater than negative control 2 times of OD value, be judged as the positive.The detected result that the mono-clonal phage antibody is combined with the A beta antigen: detect through ELISA, obtain altogether 7 positive colonies (A10, D8, F2, B9, E6, F4, G2).7 positive colony ELISA results' A value all is higher than negative control more than 2 times.2 the clone A10 and the F2 that get that ELISA wherein tires higher identify repeatedly.Result's demonstration, but these 2 the equal specific recognition A β purifying antigens of clone are not combined with the irrelevant antigen of negative control, and ELISA detection A value is higher than negative control more than 5 times (Fig. 1).

(2) PCR identifies the positive colony bacterium that obtains after the screening of positive colony bacterium liquid pcr amplification fourth round, and primer is universal primer LMB3 and Fdseq.Primer extension product is through 1% agarose gel electrophoresis analysis.The result: 7 positive bacteria performing PCR amplifications behind plasmid extraction, through gel electrophoresis, visible 7 cloned sequences are about 1050bp, meet the theoretical value (Fig. 2) of expection scFv antibody fragment PCR product.

Embodiment 3: expression and the evaluation of the anti-A β of solubility scFv fragment:

(1) positive phagemid abduction delivering in intestinal bacteria HB2151 has displaying the phage-infect logarithmic phase intestinal bacteria HB2151 of the antibody of specific combination A β, and 30min is left standstill in 37 ℃ of water-baths.Be inoculated on the SOBAG-N flat board, 30 ℃ are cultured to and grow single bacterium colony.Respectively choose 1 clone from the flat board of a plurality of different Clone Origins, be inoculated in respectively in 5ml 2 * YT-AG substratum, 30 ℃ of joltings are spent the night.The 5ml bacterium that spends the night is added in the fresh 2 * YT-AG substratum of 50ml, in 30 ℃ of jolting 1h.Centrifugal, abandon supernatant, precipitation is resuspended in the fresh 2 * YT-AI of 50ml (I:IPTG, the 1mM) substratum 30 ℃ of abduction delivering 20h.Centrifugal, supernatant filtration sterilization-20 ℃ storage.One pipe precipitation is resuspended with 1 ice-cold * TES of 0.5ml, then add 1/5 ice-cold * TES of 0.75ml and carry out vortex, ice bath 30min.Centrifugal, supernatant is the pericentral siphon extract ,-20 ℃ of storages.Another pipe precipitation is suspended with 0.5ml PBS, boil 5min, centrifugal, supernatant is full fungus extract ,-20 ℃ of storages.The whole bacterial protein of getting each clone carries out the 12%SDS-PAGE electrophoresis, as seen A10 and F2 are cloned in relative molecular mass about 33, the 000Da place presents the purpose band, and this band conforms to the molecular weight of the soluble scFv of expectation, and other 5 clones have no the purpose band (Fig. 3) of corresponding position.

(2) Western Blot: with the expression product sample preparation by the 12%SDS-PAGE electrophoretic separation after, under ice bath, 170mA, carry out electricity and turn, then nitrocellulose filter is immersed among the TBS (containing 5% skim-milk), 4 ℃ of sealings are spent the night.Again with 37 ℃ of incubation 2h of HRP mark mouse-anti c-myc tag antibody of 1: 2000.The 37 ℃ of incubation 1h of HRP mark sheep anti-mouse igg antibody that add 1: 5000 after the washing.The DAB colour developing compares with the corresponding product of inducing without IPTG.Detect the expression of the anti-A β of solubility scFv.A10 is cloned bacterium liquid supernatant, born of the same parents' pericentral siphon extract and whole bacterial protein behind the abduction delivering to carry out Western Blot and detects, as seen at relative molecular mass about 33, the 000Da place presents positive band, stronger with the whole bacterial protein reaction, this band conforms to the molecular weight of the soluble scFv of expectation, and the A10 that does not induce clone does not detect positive band (Fig. 4).

(3) ELISA detects the expression product antibody activity: step detects with the ELISA of mono-clonal phage antibody.Primary antibodie is the expression product soluble antibody, two anti-be 1: 2000 HRP mark mouse-anti c-myc tag antibody, 1: 5000 HRP mark sheep anti-mouse igg antibody of three anti-usefulness, negative control only adds 2%MPBS.Higher A10 and F2 clone's each component of tiring is expressed antibody ELISA result and is shown, antibody can be combined with people A β, be not combined with the irrelevant antigen of negative control, induce the A value of strain to be higher than and do not induce more than 2 times of strain, wherein in supernatant He in the whole bacterial protein, be higher than respectively (Fig. 5) more than 5 times, show that antibody has biologic activity.Therefore, assert that tentatively the product that A10 and F2 clone bacterium secretes is anti-human A β specificity scFv antibody.

Embodiment 4:DNA sequencing:

Extract the phagemid of positive colony, utilize ScFv dna sequencing primer, transfer to Shanghai living worker's order-checking section and through the terminal cessation method of two deoxidations the VH among the scFv DNA and VL DNA are carried out respectively sequencing.The sequential analysis of human source anti-A β specificity scFv antibody gene: obtain A10 and F2 clone's light chain, the nucleotide sequence of variable region of heavy chain, and use Blast software that sequence and the immune globulin variable region gene among the GeneBank that obtains carried out comprehensive compare of analysis.Analytical results shows: the A10 that obtains and F2 clone's variable region gene meets the scFv antibody gene, the aminoacid sequence that derivation obtains has typical antibody variable plot structure, and protein sequence is determined the initial sum termination position of CDR according to Ig blast and Kabat coding.Underscore partly is hypervariable region (HVR), is again complementary determining region (CDR), and its amino acid forms and the alterable height that puts in order.3 CDR of VH and VL form the antigen-binding site of immunoglobulin (Ig) jointly, are responsible for identification and conjugated antigen, thus the performance immunological effect.Rest part is skeleton district (FR), and its amino acid forms and the relative variation that is difficult for that puts in order.(Fig. 6), 2 clones' gene order is consistent, shows that 2 clone bacterium may derive from the original Jun grain of biting of same strain.

Wherein with the weight chain variabl area sequence called after SEQ ID NO.:1 of anti-amyloid beta antibodies gene, the protein sequence called after SEQ ID NO.:2 of its coding; With the light chain variable region sequence called after SEQ ID NO.:3 of anti-amyloid beta antibodies gene, the protein sequence called after SEQ IDNO.:4 of its coding.Wherein, the sequence of the CDR1 of variable region of heavy chain is SEQ ID NO.:5, and the sequence of CDR2 is SEQ ID NO.:6, and the sequence of CDR3 is SEQ ID NO.:7; The sequence of the CDR1 of variable region of light chain is SEQ ID NO.:8, and the sequence of CDR2 is SEQ ID NO.:9, and the sequence of CDR3 is SEQ ID NO.:10.

Embodiment 5: the prolonged preservation of engineering strain.

1) contain the TG1 bacterial strain of positive phagemid: the positive bacteriophage that utilizes display technique of bacteriophage to filter out infects the TG1 bacterium and gets.Can be at 2 * YT-AG (A:Amp, 100 μ g/ml after the recovery; G:1%Glu) go out the scFv antibody of anti-A β in the phage amalgamation and expression.

2) contain the HB2151 bacterial strain of positive phagemid: the positive bacteriophage that utilizes display technique of bacteriophage to filter out infects the HB2151 bacterium and gets.Can be at 2 * YT-AI (A:Amp, 100 μ g/ml after the recovery; I:IPTG, 1mM) in solubility expression go out the scFv antibody of anti-A β, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 12nd, 2008, preserving number is CGMCC No.2399.

Sequence table

＜110〉Chinese Academy of Medical Sciences's fundamental research institute

＜120〉variable region sequences and the encoding sequence thereof of new directed against amyloid-beta polypeptide antibody

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Gly?Leu?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met

35 40 45

Gly?Gly?Ile?Ile?Pro?Ser?Phe?Gly?Pro?Ala?Asn?Tyr?Ala?Gln?Ile?Phe

50 55 60

Gln?Gly?Arg?Val?Thr?Ile?Thr?Ala?Asp?Glu?Ser?Thr?Asn?Thr?Ala?Tyr

65 70 75 80

Met?Glu?Leu?Arg?Ser?Leu?Thr?Ser?Asp?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

85 90 95

Ala?Arg?Glu?Gly?Gly?Val?Gly?Val?Ala?Thr?Thr?Tyr?Trp?Leu?Asp?Pro

100 105 110

Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser

115 120

<210>3

<211>336

<212>DNA

＜213〉people

<400>3

cattttatgc?tgactcagcc?ccactctgtg?tcggagtctc?cggggaagac?ggtaaccatc?60

tcctgcaccc?gcagcagtgg?cagcattgcc?agcacctatg?tgcactggta?ccagcagcgc?120

ccgggcactt?cccccaccac?tgtgatctat?gaggataacc?gaagacactc?tagagtccct?180

gatcgcttct?ctggctccat?cgacagctcc?tccaactctg?cctccctcac?catctctgga?240

ctgaagactg?aggacgaggg?tgactactac?tgtcagtctt?atgatagcac?caatgcgtgg?300

gtgttcggcg?gagggaccaa?ggtcaccgtc?ctaggt 336

<210>4

<211>112

<212>PRT

＜213〉people

<400>4

His?Phe?Met?Leu?Thr?Gln?Pro?His?Ser?Val?Ser?Glu?Ser?Pro?Gly?Lys

1 5 10 15

Thr?Val?Thr?Ile?Ser?Cys?Thr?Arg?Ser?Ser?Gly?Ser?Ile?Ala?Ser?Thr

20 25 30

Tyr?Val?His?Trp?Tyr?Gln?Gln?Arg?Pro?Gly?Thr?Ser?Pro?Thr?Thr?Val

35 40 45

Ile?Tyr?Glu?Asp?Asn?Arg?Arg?His?Ser?Arg?Val?Pro?Asp?Arg?Phe?Ser

50 55 60

Gly?Ser?Ile?Asp?Ser?Ser?Ser?Asn?Ser?Ala?Ser?Leu?Thr?Ile?Ser?Gly

65 70 75 80

Leu?Lys?Thr?Glu?Asp?Glu?Gly?Asp?Tyr?Tyr?Cys?Gln?Ser?Tyr?Asp?Ser

85 90 95

Thr?Asn?Ala?Trp?Val?Phe?Gly?Gly?Gly?Thr?Lys?Val?Thr?Val?Leu?Gly

100 105 110

<210>5

<211>5

<212>PRT

＜213〉people

<400>5

Asn?Tyr?Gly?Leu?Ser

1 5

<210>6

<211>17

<212>PRT

＜213〉people

<400>6

Gly?Ile?Ile?Pro?Ser?Phe?Gly?Pro?Ala?Asn?Tyr?Ala?Gln?Ile?Phe?Gln

1 5 10 15

Gly

<210>7

<211>18

<212>PRT

＜213〉people

<400>7

Glu?Gly?Gly?Val?Gly?Val?Ala?Thr?Thr?Tyr?Trp?Leu?Asp?Pro?Trp?Gly

1 5 10 15

Gln?Gly

<210>8

<211>13

<212>PRT

＜213〉people

<400>8

Thr?Arg?Ser?Ser?Gly?Ser?Ile?Ala?Ser?Thr?Tyr?Val?His

1 5 10

<210>9

<211>7

<212>PRT

＜213〉people

<400>9

Glu?Asp?Asn?Arg?Arg?His?Ser

1 5

<210>10

<211>14

<212>PRT

＜213〉people

<400>10

Gln?Ser?Tyr?Asp?Ser?Thr?Asn?Ala?Trp?Val?Phe?Gly?Gly?Gly

1 5 10

Claims (6)

1. human source anti-A β specificity scFv antibody that comprises variable region of heavy chain, variable region of light chain and catenation sequence, it is characterized in that the aminoacid sequence of its variable region of heavy chain shown in SEQ ID NO:2, the aminoacid sequence of variable region of light chain is shown in SEQ ID NO:4.

One kind the coding human source anti-A β specificity scFv antibody as claimed in claim 1 DNA, its nucleotide sequence by the nucleotide sequence shown in the nucleotide sequence shown in the SEQ ID NO:1, the SEQ ID NO:3 and the coding catenation sequence DNA form.

3. recombinant vectors that comprises the DNA of coding as claimed in claim 2 human source anti-A β as claimed in claim 1 specificity scFv antibody.

4. recombinant host cell that comprises recombinant vectors as claimed in claim 3.

5. recombinant host cell according to claim 4, it is intestinal bacteria (Escherichia coli).

6. recombinant host cell according to claim 5, described intestinal bacteria are preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 12nd, 2008, and preserving number is CGMCC No.2399.

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