摘要
老年性痴呆(Alzheimer’s disease,AD)是一种渐进性神经退行性疾病,其病理特征是渐进性的老年斑沉积,神经突触丢失和神经纤维缠结,临床表现为记忆功能衰减及识别能力障碍,同时伴有各种神经症状和行为障碍。在老年人群,具有非常高的发病率,目前还没有特异性治疗药物。近年来AD的发病机理和药物研究方面都有突破性进展,尤其是制备针对β淀粉样蛋白(amyloid beta protein,Aβ)特异性抗体药物成为AD治疗极具价值的途径。
噬菌体展示技术(phage display)为抗体制备开创了一条简便、快速的基因工程抗体制备方法。噬菌体抗体是在噬菌体表面表达抗体分子的抗原结合片段(Antigen binding fragment,Fab)或单链抗体(single chain fragment variable, scFv),单链抗体具有分子量小,免疫原性低,穿透力强,定位迅速,易于基因工程制备和改造等优点。通过构建和筛选人源性天然噬菌体抗体库,得到Aβ寡聚体特异性人源性单链抗体,可直接应用于人AD的治疗。
利用噬菌体展示技术构建人源性天然抗体库,以可溶性Aβ1-42寡聚体对抗体库进行筛选获得针对低分子量Aβ1-42寡聚体的特异性单链抗体。分离10个健康人外周血淋巴细胞,提取总RNA,RT-PCR法得到cDNA链,以cDNA链为模板扩增得到全套人抗体VH和VL基因,经过重叠延伸PCR将VH和VL连接得到scFv片段,将scFv片段酶切后克隆至pCANTAB5E噬菌体载体,电转化TG1感受态菌,经辅助噬菌体M13K07拯救,获得库容为2.5×109单链抗体库。以可溶性Aβ1-42寡聚体为抗原,对抗体库进行四轮筛选,ELISA法筛选特异性识别Aβ1-42寡聚体的阳性克隆,将筛选到的阳性克隆B19转化至E.coli HB2151,诱导表达可溶性scFv。SDS-PAGE及Western Blot分析结果显示可溶性scFv获得了正确表达,且能够与Aβ1-42三聚体及纤维特异性结合,可溶性scFv与Aβ1-42寡聚体的亲和常数为9×10-6 mol/L。将单链抗体的重链和轻链可变区基因克隆入原核表达载体pComb3C/cFab中,转化TOP10感受态,诱导表达了重组抗体B19-Fab。SDS-PAGE及Western Blot分析结果显示,重组抗体B19-Fab得到了正确的表达。ELISA结果显示,重组抗体B19-Fab仍然保持对Aβ1-42寡聚体特异性结合活性。对单链抗体亲和力成熟进行的初步研究为后续的打下了良好的基础。本实验中获得的Aβ1-42寡聚体特异性单链抗体为老年性痴呆症(AD)的临床治疗研究奠定了基础。
Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the elder. The disease is characterized by memory impairment and cognitive obstacle. The current treatments of AD provide only symptomatic relief and are practically inefficient. Now, anti-Aβantibodies have been suggested as a potential therapeutic strategy for the treatment of AD.
Genetic engineering made it possible to generate antibody without constant region which is termed scFv(single chain fragment variable) or Fab (Antigen binding fragment). ScFv is a smaller functional unit remaining antigenic specificity and affinity. Compared with other antibodies, scFv has many advantages, smaller size, reduced immunogenicity, stronger penetrating power, rapid localization and being easily cleared up from the normal tissue. Meanwhile, scFv can be produced in large scale in bacteria or yeast and manipulated by genetic engineering. Therefore, developing scFv is of great significance in the areas of AD diagnosis and therapy.
To construct the human single-chain Fv (scFv) antibody library by phage display technology and obtain the specific scFvs (single-chain fragment variable) against soluble Aβ1-42 (Amyloid-beta ) oligomers by screening the human scFv library, peripheral blood lymphocytes (PBL)were separated from ten healthy donors, and the total RNA was extracted from the PBL. The variable heavy(VH) and variable light(VL) genes were amplified by RT-PCR and then the scFv was obtained through SOE-PCR. The scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent E.coli TG1 cells, and then a scFv phage display library containing 2.5×109clones was gained. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Aβ1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E.coli HB2151 to express soluble scFv. The soluble scFv antibodies were expressed successfully, and displayed specific binding to Aβ1-42 trimer and protofiber by SDS-PAGE and Western blot analysis.The binding affinity of soluble scFv antibodies to Aβ1-42 oligomers was 9×10-6 mol/L. For the purpose of affinity maturation, the VH and VL gene fragments were cloned into the pComb3C/cFab vector, and transformed to competent E.coli TOP10. SDS-PAGE and Western blot analysis confirmed that the soluble B19-Fab fragments were expressed successfully, and preserving the binding affinity to Aβ1-42 oligomers. The preparation of specific scFv against Aβ1-42 oligomers can be used further in the therapeutic research on AD.
引文
1徐德隆,老年与老年神经系统疾病:老年医学与保健,2002,8(3):129～130
2罗超,周江宁,刘桂建,等.微量元素与老年性痴呆,广东微量元素科学, 2004, 11(7): 1-6
3 Alpár A, Ueberham U, Brückner MK, et al. Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice. Brain Res, 2006, 1099(1): 189-98
4 Westerman MA, Cooper-Blacketer D, Mariash A, et al. The Relationship between Aβand Memory in the Tg2576 Mouse Model of Alzheimer’s Disease. J Neurosci, 2002, 22(5): 1858–1867
5 Lambert MP, Barlow AK, Chromy BA, et al. Diffusible, nonfibrillar ligands derived from Abeta 1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci USA, 1998, 95(11): 6448-6453
6 Klein WL, Stine WB Jr, Teplow DB. Small assemblies of unmodified amyloidβ-protein are the proximate neurotoxin in Alzheimer’s disease. Neurobiol Aging, 2004, 25(5): 569–580
7 Cirrito JR, May PC, O'Dell MA, et al. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-βmetabolism and half-life. J Neurosci, 2003, 23(26):8844–8853
8 DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-Aβantibody alters CNS and plasma Aβclearance and decreases brain Aβburden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci. USA , 2001 , 98(15): 8850-8855
9 Bard F.C, Cannon R, Barbour R.L. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med, 2000, 6(8): 916-919
10 Bacskai BJ, Kajdasz ST, McLellan ME, et al. Non-Fc-Mediated Mechanisms Are Involved in Clearance of Amyloid-βIn Vivo by Immunotherapy. J Neurosci, 2002, 22(18): 7873–7878
11 Solomon B, Koppel R, Hanan E, et al. Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimerβ-amyloid peptide. Neurobiology, 1996, 93(1): 452-455
12 Arbel M, Solomon B. Immunotherapy for Alzheimer’s disease: attacking amyloid-βfrom the inside. Trends Immunol, 2007, 28(12): 511-513
13 Horikoshi Y, Mori T, Maeda M, et al. AβN-terminal-end specific antibody reducedβ-amyloid in Alzheimer model mice. Biochem Bioph Res Co, 2004, 325(2): 384–387
14 Schenk D, Barbour R, Dunn W, et al. Immunization with amyloid-b attenuatesAlzheimer disease-like pathology in the PDAPP mouse. Nature, 1999, 400:173-177
15 Hock C, Konietzko U, Papassotiropoulos A, et al . Generation of antibodies specific forβ-amyloid by vaccination of patients with Alzheimer disease. Nature Medicine, 2002, 8(11): 1270 - 1275
16 Orgogozo JM, Gilman S, Dartigues JF, et al. Subacute meningoencephalitis in a subset of patients with AD after Aβ42 immunization. Neurology, 2003, 61: 46–54
17李旺,田珂,王月丹.阿尔茨海默氏病的免疫治疗回顾.中国科技论文在线
18 Dodel RC, Du Y, Depboylu C, et al. Intravenous immunoglobulins containing antibodies againstβ-amyloid for the treatment of Alzheimer’s disease. J Neurol Neurosurg Psychiatry, 2004, 75(10): 1472-1474
19 Cheryl A. Hawkes, JoAnne McLaurin. Clinical immunotherapy trials in Alzheimer’s disease. Drug Discovery Today: Therapeutic Strategies, 2008, 5(3): 177-183
20 Touchon J, Vellas B, Katchaturian Z. Editorial: CTAD International Research Conference: Clinical Trials in Alzheimer's Disease. J Nutr Health Aging, 2009,13(3): 204
21 Hawkes CA, McLaurin J. Immunotherapy as treatment for Alzheimer's disease. Expert Rev Neurother, 2007, 7(11): 1535-1538
22 Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, 1985, 228(4705):1315-1317
23 McCafferty J, Griffiths AD, Winter G. Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 1990, 348(6301): 552-524
24 Orlandi R, Güssow DH, Jones PT, et al. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Biotechnology, 1992, 24: 527-531
25 Horwitz AH, Chang CP, Better M, et al. Secretion of functional antibody and Fab fragment from yeast cells. Proc Natl Acad Sci. U S A, 1988, 85(22): 8678-8682
26 Skerra A, Plückthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science, 1988, 240(4855): 1038-1041
27 McCafferty J, Griffiths AD, Winter G, et al. Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 1990, 348(6301):552-554
28 Knappik A, Ge L, Honegger A, Pack P, et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J Mol Biol, 2000, 296(1): 57-86
29 Büssow K, Konthur Z, Lueking A, et al. Protein array technology. Potential use in medical diagnostics. Am J Pharmacogenomics, 2001, 1(1): 37-43
30 Alter G. High-throughput protein arrays: prospects for molecular diagnostics. TrendsMol. Med, 2002, 8: 250-253
31 Lueking A, Horn M, Eickhoff H, Protein microarrays for gene expression and antibody screening. Anal Biochem, 1999, 270(1): 103-111
32 Dalton R, Abbott A. Can researchers find recipe for proteins and chips? Nature, 1999, 402(6763): 718-719
33 de Wildt RM, Mundy CR, Gorick BD, et al. Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat Biotechnol, 2000, 18(9): 989-994
34 Cleary JP, Walsh DM, Hofmeister JJ. Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function. Nat Neurosci, 2005, 8(1): 79-84
35 Townsend M, Shankar GM, Mehta T, et al. Effects of secreted oligomers of amyloid beta-protein on hippocampal synaptic plasticity: a potent role for trimers. J Physiol, 2006, 572( 2): 477-492
36 Birmingham K, Frantz S. Set back to Alzheimer vaccine studies. Nat Med, 2002, 8(3): 199-200
37 Orgogozo JM, Gilman S, Dartigues JF. Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization. Neurology, 2003, 61(1): 46-54
38王尚资.全合成人源抗体库和人源化改型抗体库的构建及筛选.中国海洋大学, 2004-11-02
39 Sblattero D, Bradbury A. A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology, 1998, 3(4): 271-278
40 Marks JD, Tristem M, Karpas A. Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes. Eur J Immunol, 1991, 21(4): 985-991
41 Winter G, Griffiths AD, Hawkins RE. Making antibodies by phage display technology. Annu Rev Immunol, 1994, 12: 433-455
42 Lambert MP, Viola KL, Chromy BA, et al. Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies. J Neurochem, 2001, 79(3): 595-605
43 Klein WL. Abeta toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets. Neurochem Int, 2002 , 41(5): 345-352
44 Cribbs DH, Ghochikyan A, Vasilevko V, et al. Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol, 2003, 15(4): 505-514
45 Asuni AA, Boutajangout A, Scholtzova H. Vaccination of Alzheimer's model mice with Abeta derivative in alum adjuvant reduces Abeta burden without microhemorrhages. Eur J Neurosci, 2006, 24(9): 2530-2542
46 Lambert MP, Velasco PT, Chang L, et al. Monoclonal antibodies that target pathological assemblies of Aβ. J Neurochem, 2007, 100(1): 23-35
47 Lacor PN, Buniel MC, Chang L. Synaptic targeting by Alzheimer's-related amyloid beta oligomers. J Neurosci, 2004 , 24(45): 10191-10200
48 Lee EB, Leng LZ, Zhang B, et al. Targeting amyloid-βpeptide (Aβ) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Aβprecursor protein (APP) transgenic mice. J Biochem, 2006, 281(7): 4292–4299
49 Le Gall F, Kipriyanov SM, Moldenhauer G, et al. Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding. FEBS Lett, 1999 453(1-2): 164-168
50 Smith GP. Surface display and peptide libraries. Gene, 1993, 128(1): 1-144
51 Sblattero D, Bradbury A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol, 2000, 18(1): 75-80
52 Model P, Russel M. Filamentous bacteriophage. The Bacteriophage, 1988, 2: 375-456
53 Bai Y, Feng H. Selection of stably folded proteins by phage-display with proteolysis. Eur J Biochem, 2004, 271(9): 1609-1614
54 Cheng LS, Liu AP, Yang JH, et al. Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185(c-erbB-2). Cell Res, 2003, 13(1): 35-48
55 Ward ES, Güssow D, Griffiths AD, et al. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature, 1989, 341(6242):544-546
56 Marks JD, Hoogenboom HR, Bonnert TP, et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol, 1991, 222(3): 581-597
57 Hughes-Jones NC, Bye JM, Gorick BD, et al. Synthesis of Rh Fv phage-antibodies using VH and VL germline genes. Br J Haematol, 1999, 105(3): 811-816
58 Friguet B, Chaffotte AF, Djavadi-Ohaniance L, et al. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods, 1985 , 77(2): 305-319
59 Winter G, Griffiths AD, Hawkins RE, et al. Making antibodies by phage display technology. Annu Rev Immunol, 1994, 12: 433-455
60 Barban V, Fraysse-Corgier S, Paranhos-Baccala G, et al. Identification of a human epitope in hepatitis C virus (HCV) core protein using a molecularly cloned antibodyrepertoire from a non-symptomatic, anti-HCV-positive patient. J Gen Virol, 2000, 81(2): 461-469
61 Dong L, Chen S, Richter M, et al. Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (Close Homolog of L1) enhance neurite outgrowth. J Neuroscience Research, 2002, 69: 437-447
62 Steinberger P, Sutton JK, Rader C, et al. Generation and characterization of a recombinant human CCR5-specific antibody. J Biol Chem, 2000, 275(46): 36073-36078
63王刚,王琰.硫氰酸盐洗脱法测定噬菌体抗体的相对亲和力.中华微生物学和免疫学杂志, 2000, 20(4): 355-357
64 Schaaper RM. Mechanisms of mutagenesis in the Escherichia coli mutator mutD5: role of DNA mismatch repair. Proc Natl Acad Sci. USA,1988, 85(21): 8126-8130
65 Fromant M, Blanquet S, Plateau P. Direct random mutagenesis of gene-sized DNA fragments using polymerase chain reaction. Anal Biochem, 1995, 224(1): 347-353
66 Arnold FH, Wintrode PL, Miyazaki K. How enzymes adapt: lessons from directed evolution. Trends Biochem Sci, 2001, 26(2): 100-106
67 Low NM, Holliger PH, Winter G. Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mol Biol, 1996, 260(3): 359-368
68 Jermutus L, Honegger A, Schwesinger F. Tailoring in vitro evolution for protein affinity or stability. Proc Natl Acad Sci. USA, 2001, 98(1): 75-80
69 Boder ET, Midelfort KS, Wittrup KD. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci. USA, 2000, 97(20): 10701-10705
70陈志南,边惠洁,蒋建利.肝癌单抗研究应用现状与展望.华人消化杂志, 1998, 6: 461-462
71骞爱荣,商澎,李郁,等. HAb18G/CD147拮抗肽抗肝癌转移作用的体外实验.世界华人消化杂, 2003, 11: 255-259
72朱进,赵萍,焦永军,等.可内化的人源抗Met基因工程抗体Fab的亲和力成熟与特性分析.生物化学与生物物理进展. 2007, 34(1):73-79
73袁斌,侯晓军,王慧,等.构建轻链次级库提高人抗TNF—α抗体的亲和力.细胞与分子免疫学杂志, 2001, 17(2): 179-181