摘要
乳腺癌是女性常见的恶性肿瘤之一，发病率正呈逐年上升趋势，这一现象在大中城市尤为突出。目前，乳腺癌治疗的主要手段仍然是手术治疗，但仅通过手术不可能根治乳腺癌。在手术治疗的基础上辅以化疗、放疗、免疫治疗、中医中药治疗等各项治疗手段，才能取得一个较好的治疗效果。多年来，人们一直在探索应用免疫学的方法来治疗乳腺癌，但直到1998年，美国FDA批准针对乳腺癌基因过表达产物HER2受体的人源化单克隆抗体Herceptin用于乳腺癌的临床治疗，人们才真正看到了免疫治疗乳腺癌的曙光。
传统的杂交瘤技术制备单克隆抗体费时费力。噬菌体抗体库技术的出现使抗体的制备产生了突破性的进展。该技术与传统的杂交瘤技术相比，具有简便易行；筛选容量大，过程短；利于进一步进行基因工程改造；生产成本低等优点。噬菌体展示的抗体片段可以是单链可变区片断（scFv）或Fab段，也可以是用一段多肽链或二硫键连接的双抗体或多抗体片段。
ScFv是由免疫球蛋白的重链可变区(VH)和轻链可变区(VL)通过一段连接肽连接而成的重组蛋白，是具有完全抗原结合位点的最小抗体片段，大小为完整抗体的六分之一，分子量约为27KD。ScFv弥补了传统单克隆抗体抗原性强、穿透力弱等不足，在导向治疗方面有着巨大的应用潜力。虽然噬菌体抗体库技术应用的时间较短，但在肿瘤的诊断和治疗、抗原表位分析、药物设计等多个领域显示出了巨大的发展潜能和广阔的应用前景。本研究应用噬菌体展示技术构建了一个抗乳腺癌细胞的噬菌体单链抗体
库，旨在筛选出与人乳腺癌细胞特异结合的单链抗体，以期有助于乳腺癌的导向诊治。
首先用人的乳腺癌细胞MCF-7免疫BALB/c小鼠，从MCF-7乳腺癌细胞免疫的BALB/c小鼠脾脏提取总RNA，设计简并引物，RT-PCR扩增小鼠抗体重、轻链可变区基因，用(Gly4Ser)3连接肽基因，经重叠延伸反应，在体外将VH和VL连接成scFv。scFv回收纯化，用SfiⅠ、NotⅠ进行酶切，与经同样双酶切反应的表达载体pCANTAB5E连接，电转化大肠杆菌TG1，并经辅助噬菌体M13KO7超感染，scFv展示在重组噬菌体表面，构建噬菌体单链抗体库。扩增的VH大小为340bp，VL大小为320bp左右，scFv基因大小为750bp左右。构建了库容为1.2×106的抗乳腺癌细胞的噬菌体单链抗体库。以MCF-7乳腺癌细胞为靶标对抗体库进行吸附、洗脱、扩增”的富集筛选，ELISA法鉴定各单克隆与乳腺癌MCF-7细胞、肝癌HepG2细胞、宫颈癌Hela细胞的结合活性。经过5轮筛选，从中筛选出一株与人乳腺癌细胞系MCF-7特异性结合较的噬菌体单链抗体phage scFv-507。经序列分析，此基因全长732bp，编码244个氨基酸序列，通过Blast检索，此scFv重链与轻链可变区基因与鼠的IgG同源性达到95%以上。
将重组的scFv-507转化非琥珀抑制的菌株E.coli TOP10，IPTG进行诱导表达，多肽链的翻译终止于E-tag，scFv-E-tag在信号肽引导下分泌到壁膜间隙，成为可溶性的scFv-E-tag融合蛋白，可以用抗E-tag抗体来检测scFv的表达情况。诱导表达的菌液作SDS-PAGE和Western blot分析，发现在相对分子量32kDa处有一条清晰的显色条带，为能与anti-E tag抗体特异性结合的pⅢ signal-scFv-E tag，与文献报道相符，说明scFv-507在E.coli TOP10得到了正确表达。
为深入研究特定单链抗体(scFv)的功能，将识别MCF-7乳腺癌细胞的
单链抗体scFv-507克隆入原核表达载体pET24a，构建高效表达载体pET-24a-scFv-507，转化E.coli BL21(DE3)，IPTG诱导表达，scFv-507基因表达产物在大肠杆菌以不溶性包涵体形式高效表达。超声破碎细菌细胞得包涵体，经2mol/L 尿素洗涤后，6mol/L盐酸胍溶解包涵体，利用其产物末端携带6个连接的组氨酸标签与金属螯合层析介质中的镍离子的亲和性，应用Ni-NTA金属螯合层析在蛋白质变性条件下进行纯化，纯化蛋白细胞直接稀释复性，探索复性条件(温度、时间、GSSG及GSH的浓度等)。SDS-PAGE分析蛋白的纯度，ELISA检测复性蛋白的活性。诱导表达的scFv占全菌蛋白的20%，变性纯化的蛋白在5mM氧化型谷胱甘肽（GSSG），0.5mM的还原型谷胱甘肽（GSH），400mM L-Arg条件下稀释复性，复性的scFv纯度大于90%，浓度达到75μg/mL，复性率达到50%。ELISA检测纯化复性后的scFv-507活性。实验结果表明scFv可与乳腺癌MCF7细胞、231细胞有效结合，而与正常细胞系L02则呈阴性反应。
本研究获得的与人乳腺癌细胞系MCF-7结合特异性结合的单链抗体为进一步进行乳腺癌导向治疗的研究奠定了基础。
In recent years monoclonal antibodies(MAbs) have become accepted tools for the detection and treatment of breast cancer.A humanized anti-HER2 antibody (Herceptin) has been approved by the United States Federal Drag Administration for the treatment of breast cancer. The successes with antitumor antibodies in patients has led to renewed interest in the identification of novel tumor-associated antigens suitable for antibody targeting.
Until recently, the production of antibodied was limited to very laborious and time-consuming processes involving animal immunization schemes and/or hybridoma generation. Phage display antibody library has now made it possible to clone antibody genes in bacteria. Such of antibody genes makes it technically feasible to produce antibodies quickly in bacterial cultures and to genetically manipulate their structure such that ultimately, antibodies to any antigen may be created. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. One of the most successful approaches is to display single-chain Fv(scFv)antibodies on filamentous phage.
scFv is an antigen-binding protein, composed of an immunoglobulin heavy-chain variable domain(VH) and a light-chain variable domain(VL) joined together by a flexible peptide linker. scFv is the smallest fragment that
maintains the binding specificity and affinity of the entire antibody. Because of their small size, scFv fragment could be useful in tumor imaging and therapeutic strategies. Together with function-based selection procedure, antibody phage display technologies would open more challenging applications and provide a powerful tool for drug and target discovery. In this study, we constructed a phage display single-chain variable fragment(scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the phage display library.
The total RNA was isolated from spleens of BALB/C mice immunized with breast cancer cell line MCF-7. A set of degenerate oligonucleotide primers were designed and used to amplify VH and VL genes which were then joined into single chain variable fragment(scFv) by a DNA linker encoding peptide(Gly4Ser)3. The scFv fragments were cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 protein in E. coli TG1.The recombinant phage were rescued by being cocultured with helper phage M13KO7 .The scFv fusion protein was displayed on the surfaces of recombinant phages.Amplification of VH generated a DNA fragment with the expected length(about 340bp),while VL generated an expected 320bp fragment. The scFv DNA fragment is about 750bp.A repertoire of 1.2×106 clone phage display antibody library was constructed. The library was screened with MCF-7 cells by binding-elution-enrichment procedure.The positive recombinant phages were identified by ELISA with MCF-7 cells ,HepG2 cells and Hela cells. After five rounds of panning, a phage-scFv named phage scFv-507 binding MCF-7cells specifically was selected from the library.DNA sequence analysis showed scFv-507 DNA had
732bp and encoded 244 amino acid.
The recombinant scFv-507 was transformed into nonsuppression E.coli Top10 that was allowed to express soluble scFv proteins by induction of IPTG. After induced by IPTG, the amber stop codon could be recognized and scFv-507-E tag fusion protein secreted into the periplasm of bacteria E.coli Top10. The E-tag at the C-terminus allowed the protein to be detected by Western blot. The relative molecular mass of expressed scFv-507 was about 32KD showed by SDS-PAGE and Western blot, which was consistent with expected molecular weight.
To increase the yield of scFv-507in E.coli, the scFv-507 gene was transferred to an expression vector pET24a.The recombinant expression vector was transformed into E.coli BL21(DE3).After induced by IPTG,scFv-507 recombinant protein was expressed efficiently as inclusion bodi
引文
[1].郑莹,李德录,向泳梅,等.上海市区乳腺癌流行现状及趋势分析.外科理论与实践,2001,6:219 221
[2]. 戴朝六,余云,刘思卿,等.关于日本乳腺癌保留乳房治疗问题[Ｊ].中国实用外科杂志,1996,9:567-569.
[3]. Veronesi V, Salvadori B, Luini A, et al. Conservetive treatment of early Breast cancer.[J] Ann Surg,1990,211(3):250-253
[4]. Levitt S H. Is there a role for post-operative adjuvant radiation in breast cancer.[J].Int J Radiat Oncol Biol Phys,1988,14:787
[5]. Host H, Brennbovd I O, Loed M. Postoperative radiotherapy in breast cancer.[J] Int J Radiat Oncol Biol Phys,1986,12:727
[6].Robert N J.Clinical efficacy of tamoxifen.Oncology(Huntingt),1997,11:
15-20
[7]. Carbonepp. Breast cancer adjuvant therapy[J].Cancer,1990,66:1378
[8]. 王莉,雷秋模.乳腺增生与乳腺癌[Ｍ].北京:中国科学技术出版社,1993.
[9]. Anonym Oul. New aromatase inhibitor for breast cancer. Drug Ther B ull,1997,35:55-56
[10].Byrne M J. Gebsk V, Foroes J, et al. Medroxyprogesterone accurate addition or substitution for tamoxifen in advanced tamoxifen-resistant breast cancer a phase Ⅲ randomized trial. Australian New Zealand Breast Cancer Trials Group. [J] Clin Oncol,1997,15(a):3191-3198
[11].Carter P, Presta L, Gorman CM et al. Humanization of an anti p185HER2 antibody for human cancer therapy[J].Proc Natl Acad Sci USA 1992,89: 4285-4289
[12].Schintt S J. Breast cancer in 21st century: new opportunities and new challenges[J].Mod Pathol,2001,14(3):133-146.
[13].Stebbing J, Copson E, O’Reilly S. Herceptin(trastuzamab) in advanced breast cancer.[J].Cancer Treat Rev,2000, 26(4):287-294
[14].Horton J. Trastuzumab use in breast cancer:clinical issues.[J]Cancer Control,. 2002,9(6):499-507.
[15]. McCafferty J, Griffiths AD, winter G, et al. Phage antibodies:filamentous phage displaying antibody variable domains. [J]Nature.1990;348:552-554.
[16]Yokota T，Milenic DE, Whitlow M et al. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms[J].Cancer Reasearch,1992,52:3402-3408.
[17]. Sapats SI,Heine HG,Trinidad L,et al. Generation of chicken single chain antibody variable fragments(scFv) that differentiate and neutralize infectious bursal disease virus(IBDV).[J] Arch Virol.2003;148(3):497-515
[18]. Huston JS, Levinson, Mudgett-Hunter M, et al. Protein engineering of antibody binding sites:Recovery of specific activity in an ani-digoxin single-chain Fv analogue produced in Eschenchia coli. Proc Natl Acad Sci USA,1988; 85:5879-5883.
[19].Hennecke F,Krebber C,Pluckthun A. et al.Non-repetitive single-chain Fv linkers selected by selectively infective phage(SIP) technology.[J] 1998;11:405-410
[20].Zdanov A, Li Y, Bundle DR, et al. Structure of a single-chain antibody variable domain(Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution.[J] Proc Natl Acad Sci,1994,91(14):6423-6427
[21].Kontermann RE.Liu Z, Schμlze RA,et al. Characterization of the epitope recognized by a monoclonal antibody directed against the largest subunit of Drosophila RNA polymerase II.[J]Chem Hoppe-Seyler,1995;376:437
[22].George A J, Jamar F, Tai M S et al. Radiometal labeling of recombinant
proteins by a geneticall engineering minimal chelation site:Technetium-99m coorination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide[J]Proc Natl Acad Sci USA,1995;92(18):83568-8362
[23].Kreitman RJ, Bailon P, Chaudhary VK,et al.Recombinant immunltoxins containing anti-Tac(Fv) and derivatives of Pseudomonas exotoxin produce complete regression inMice of an interleukin-2 receptor-expressing human carcinoma.[J] Blood.1994,83(2):426-434
[24].Benhar J, Rerter y, Pai LH, et al. Administration of disμlfide-stabilized Fv-immunotoxin B1(dsFv)-PE38 and B3(dsFv)-PE38 by curing large tumor xenografts in nude mice.Int J Cancer,1995,62:351-355
[25].Pavlinkova G, Booth BJ, Batra SK, et al. Radio immunotherapy of human colon cancer xenografts using a dimeric single-chain Fv antibody constructs[J] Clin Cancer Res,1999,5(9):2613-2619
[26].Di Paolo C, Willuda J,Kubetzko S, et al. A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecμle-specific single-chain antibody Fragment has potent and selective anti-tumor activity. [J].Cin Cancer Res.2003,9(7):2837--2848
[27].Hombach A, Schneider C, Sent D, et al. An entirely humanized CD3 zeta-chain signaling receptor that directs peripheral blood T cells to specific lysis of carcinoem bryonic anrigen-positive tumor cells [J].Int J Cancer,2000,88(1):115-120
[28].Heuser C,Ganser M,Hombach A,et al.An anti-MUC1-antibody-interl eukin-2 fusion protein that activates resting NK cells to lysis of MUC1 positive tumor cells ,[J]Br J cancer.2003,89(6):1130-1139
[29].Ma J, Lou D, Kwon GS, et al. Use of encapsμlated single chain antibodies for induction of anti-idotypic humoral and cellμlar immune responses [J].J Pharm Sci, 1998;87(11):1375-1378
[30].Ma J, Li Z, Lou D. single chain antibody vaccination in mice against human ovarian cancer enhanced by microspheres and cytokines.[J].J Drug
Target,2003,11(3):169-176
[31].Spellerberg MB, Zhu D,Thomsett A, et al. DNA vaccination against lymphoma promotion of anti-idiotypic antibody responses induced by single chain Fv genes by fusion to tetanus toxin fragment C.[J]J Immunol.1997;159(4):1885-1892
[32].Jean D, Tellez C, Huang S, et al. Inhibition of tumor growth and metastasis of Human melanoma by intracellμlar anti-ATF-1 single chain Fv fragment.[J]. Oncogene,2000,19(22):2721-2730
[33].Khare PD, Liao S, Hirose Y, et al. Tumor growth suppression by a retroviral vector displaying scFv antibody to CEA and carrying the iNOS gene.[J] Anticancer Res.2002,22(4):2443-2446
[34]Duan L, Bagasra O, Laughlin MA, et al. Potent inhibition of human immunodefic-iency virus type I replication by an intracellμlar anti-Rev single-chain antibody.[J] Proc Natl Acad Sci USA.1994.91:5075-5079
[35].Jian W, Venugopai K, Goμld EA. Intracellμlar interference of Tick-Borne Flavivirus Infection by Using a single-chain antibody fragment delivered by recombinant sindbis virus. Journal of Virology,1995,69(2):1044-1049
[36].Bouhamdan M, Strayer DS, Wei D, et al. Inhibition of HIV-1 infection by down-regμlation of the CXCR4 co-recepter using an intracellμlar single chain variable fragment against CXCR4.[J]Gene Ther.2001,8(5):408-418
[37].Newsred WJ, Ramjeesingh M, Zywμlko M, et al. engineering resistance to trypsin inactivation into l-asparaginase through the production of a chimeric
[38].Tsurushita N, Fu H and Warren C. Phage display vectors for in vivo recombination of immunoglobμlin heavy and light chain genes to make large combinatorial libraries[J] Gene 1996;172:59- 63.
[39].Clackson T, Hoogenboom H R, Griffiths AD, et al. Making antibody fragments using phage display libraries. Nature，1991，352（15）:624-628.
[40]Olandi R , Gussow D ,Jones PT, et al. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction[j].Proc.Natl.Acad.Sci.USA 1989，86:3833-3837.
[41].Qiao-ping Yuan,Clarke J R,Hui-ren Zhou, et al. Molecular Cloning, Expression,and Characterization of a Functional Single-chain Fv Antibody to the Mycotoxin zearalenone[J].Applied And Environmental Microbiology,1997,63（1）:263-269.
[42]. Kang AS,Barbas CS,Janda KD, et al. Linkage of recognition and replication functions by assembling combinatorial antibody Fab library along phage
[43].Adams GP, McCartney JE, Tai MS, et al. Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-cerB-2 single-chain Fv. Cancer Research, 1993; 53:4026-4034.
[44].Hoogenboom H R, De Bruine A P, Hufton S E, et al. Antibody phage display technology and its applications[J].Immuno,1998,4:1-20
[45].Griffiths A D, Duncan A R. Strategies for selection of antibodies by phage display [J].Curr Opin Biotechnol,1998,9:102-108
[46].Chowdhury P S, Chang K, Pastan I. Isolation of antimesothelin antibodies from a phage display library[J] .Mol Immunol,1997,84:9-20
[47].陏建华，宋增璇，佘鸣等.KGla细胞免疫小鼠脾细胞scFv噬菌体展示文库的构建及其表达.[J]中国免疫学杂志，2000，16(6):318-321
[48].Harrd H，Henderikx P，Hoogenboom H R.Creating and engeering human antibodies for immunotherapy.Advanced Drug Del Rev.1998,31:5-31
[49].Pasqualini R, Rouslahti E, Orgen targeting in vivo using phage display peptide libraries. [J]. Nature ,1996;380:364-366
[50].Arap W, Pasqualini R, Rouslahti E. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science, 1998; 279:377-380
[51]. Ridgway JB, Ng E, Kern JA, et al. Identification of a human anti-CD55
single-chain Fv by subtractive panning of a Phage library using tumor and nontumor cell lines[J].Cancer Research,1999,59:2718-2723.
[52]. 张广发，王琰，王雅明等.噬菌体抗体库的构建及抗肿瘤单抗的筛选。中华肿瘤杂志，1995，17（4）：258-262
[53].Dranoff G,Jaffee E,Lazenby A,et al.Vaccination with irradiated tumor cells engineered to secret murine gramμlocyte macrophage colony stimμlating factor stimμlates potent,specific ,and long lasting anti tumor immunity.proc Natl acad Sci USA,1993,90(8):3539-3543
[54].Guise AD，West S M，Chaudhuri J B. Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies.Mol Biotech, 1996,6(1):53～64
[55].Dabora J M, Sanyal G, Middaugh C R..Effect of polyanion on the refolding of human acidic fibroblast growth factor.[J].Biol Chem, 1991,266(35):23637～23640
[56].Tsumoto K,Shinoki K, Kondo H, et al.Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment.[J] Immuno Meth,1998,219:119-129
[57].Sambrook J, Russell D.分子克隆实验指南，2002年第三版.
[58]司徒镇强，吴军正.细胞培养，1996年第一版.
[59]. Parsons HL,Earnshaw JC,Wilton J, et al .Directing phage selections towards specific epitopes. [J].Protein Engineering,1996;9(11)1043-9
[60]. Hemminki A,Niemi S,Hoffren AM, et al.Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection. [J].Protein Engineering,1998;11(4):311-319
