抗人卵巢癌scFv基因的构建、克隆、筛选和表达
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摘要
卵巢癌是妇科恶性肿瘤中最主要死亡原因之一。尽管近十年来，随着肿瘤细胞减灭术和化疗水平的提高，卵巢癌的生存率有所提高，但由于大多数病人发现时已是晚期，所以生存率一直很差，其5年生存率在30％左右，因此探索新的治疗方法非常必要。
自从杂交瘤技术问世以来，单克隆抗体在人类肿瘤诊断和治疗上的应用成为人们关注的焦点。然而，单克隆抗体在肿瘤诊断治疗上所取得的成就并不大，因为抗体的应用有很高的要求。例如，治疗性的或用于体内诊断的抗体片段要求肿瘤穿透力强，在血液中清除更快、免疫原性低等。同时，还需要一个更有效的方法，可以很容易得到对每一个抗原都有优惠特性和有效作用的分子，并达到高水平产量，以满足抗体应用的高标准和日益增加的应用量。由于大多数单克隆抗体是鼠源的，且体积相对较大，故其作为治疗和诊断试剂的主要限制是肿瘤穿透力较差及其免疫原性。此外，直到最近，单克隆抗体的产量依旧受限于涉及到动物免疫和／或杂交瘤制备这一非常耗时耗力的过程。单链抗体片段(scFv)因此成为一种特别流行的抗体形式。该分子的体积减小只有抗原结合部分，由抗体重链可变区(VH)和轻链可变区(VL)经过一个弹性linker连接而成。与完整抗体IgG及抗体Fab段相比，scFv片
第四军医大学博士学位论文 复6页
段相对小的体积和没有k段，使其能更快穿透肿瘤，在血液中更快清除、免
疫原性低，且能很容易的用来构建含有药物或细胞因子的嵌合分子、双价或
双特异抗体。此外鼠s。Fv可通过基因工程的方法修饰他们的亲和性或将鼠的
框架置换为人的来进一步提高他们在人体内的作用，而且大多数ScFv片段
能很容易的在大肠杆菌中重组表达获得足够的量。因此S。FV成为将药物、
毒素、放射性毒素导向肿瘤的很有价值的分子，在治疗和诊断试剂的发展上
具有巨大的潜力。
目前，成功生产重组抗体的方法已研制出来，此方法依赖于将抗体片段
以融合蛋白呈现在噬菌体表面的噬菌体呈现系统。由于呈现的抗体保持着它
的抗原结合活性，因此可通过亲和筛选富集表达特异性抗体的重组噬菌体。
使用这种方法，具有特异性和亲和活性的抗体便能从富集重组噬菌体中筛选
出来。重组噬菌体技术能有效的模仿免疫多样性和选择性的特征，能很容易
的通过测序、突变和筛选来提高重组噬菌体抗体对抗原的结合力，并可用细
菌系统来代替目前的动物免疫和杂交瘤制备，无限量的合成和表达实际上可
针对任何抗原的抗体分子。本研究的目的就是利用噬菌体表面呈现技术构建
一个较大容量的抗人卵巢癌单链抗体基因文库，并表达出活性片段，以期更
早应用于临床，对卵巢癌的诊断、治疗水平的提高有所帮助。
由于上皮性卵巢癌在卵巢癌中致死率中一直最高，因此我们选用来自上
皮性卵巢癌的细胞株HO七，SKOV3，COCI和组织做抗原免疫6七周的
Balb／c小鼠从免疫小鼠脾脏中分离tnRNA，RTPcR分别扩增抗体重、轻链
可变区基因，二者经一个特殊构建的 Linkef（Gly4Sef）连接形成 scFv，然后
克隆到高效噬菌粒载体pCANTABSE中，并在大肠杆菌中表达。经M13KO7
辅助噬菌体挽救后，产生呈现抗体sCFv片段的重组噬菌体。获得库容量约为
4．5 xl 06噬菌体 ScFv库，经过四轮 palllling淘筛后，能与特异性抗原结合的噬
菌体呈现的抗体被筛选出来和富集，噬菌体呈现的重组抗体用ELISA进行检
测和鉴别。结果显示：从随机挑取的 94个噬菌体克隆中筛选到 31个具有抗
DePGrtfllBltt ofobsW and GynecolbglL Xi JwHoWI，m－ FMMU
g
纷四军医大学博士学位论文 第7更
原结合活性的阳性克隆，所有的阳性克隆与其他肿瘤细胞、人正常成纤维细
胞进行ELISA检测，无阳性反应。说明通过pannillg淘筛，从构建的噬菌体
抗体文库中成功去除了能与其他肿瘤细胞和人正常细胞共同结合的噬菌体抗
体组分，获得的阳性克隆具有卵巢癌细胞特异性。
由于可溶性重组抗体能快而经济的生产，是非常有用的免疫试剂，因此
抗原阳性的克隆被分离出来后，我们在大肠杆菌中进行可溶性单链抗体片段
的表达，并用能识别 E－tag sk段的 ntiE Ta gtR以验证。可溶性抗体在大肠杆
菌 TG和 HBZI sl细胞中都能产生，但在 HBZISI细胞中的产量较高，因此
我们用抗原阳性的噬菌体感染 HBZISI细胞，加诱导剂 IPTG诱导可溶性
SCFV蛋白的产生。可溶性抗体可能位于培养上清、细菌周质和细菌内，离心
沉淀细胞，制备不同细胞提取物，用 SDS－PAGE和 nti＊Ang进行 Western blot
检测加以证实可溶性抗体集中的位置。结果显示：表达蛋白为分子量～30KDa
的可溶性蛋白，与预期大小相符，证实有可溶性抗体产生，可溶性抗体主要
集中在细菌内和细菌周质中，但只有细菌周质中细胞内的可溶性抗体具有生
物功能活性。表达的重组可溶性抗体继续用 D。t hi。t、流式细胞仪和免疫荧
光竟争抑制实验检测其与抗原的结合活性。结果显示?
Ovarian carcinoma is the leading cause of death from gynecological cancer. Although advances in treatment with cytoreductive surgery and chemotherapy have improved the survial rate in the last decade, in most patients,the disease is diagnosis at an advanced stage,survial is still poor,with a 5-yr survial rate of only 30%. thus requiring new therapeutic modalities.
Since the advent of hybridoma technology, the potential of murine mAbs for therapeutic and diagnostic application in human cancers has been a focus of interest. However, the therapeutic use of monoclonal antibodies in human cancers has emt with limited success, because there are great demands on such applications of antibody fragments. For example, therapeutic antibody fragments require lower immunogenicity, exhibit more rapid blood clearance and better tumor penetration.The high demand for and the increasing number of applications of antibodies require more efficient methods for their high-level production and the availability of molecules with favorable properties for every antigen. As most of McAb are rodent antibodies with relatively large size, The major limitations of monoclonal antibody conjugates as therapeutic agents have
been inadequate tumor penetration and immunogenicity. Furthermore, until recently, the production of antibodies was limited to very laborious and time-consuming processes involving animal immunization schemes and/or hybridoma generation. Thus single-chain Fv (scFv) fragments became a particularly popular antibody format. The size of these molecules is reduced to the antigen-binding part of the antibody, and they contain the variable domains of the heavy and light chain connected via a flexible linker. Compared to intact IgG or Fab fragments, their relatively small size and absence of Fc-effector domains may allow them to penetrate tumors more rapidly, exhibit more rapid blood clearance and lower immunogenicity and can readily be used to construct chimeric molecules containing drus or cytokines or bivalent and bispecific antibodies, In addition, Murine scFv can be also engineered for improving their performances in vivo in humans by modulating their affinity or by replacing murine by human frameworks and Most scFv fragments can be obtained easily from recombinant expression in Escherichia coll in sufficient amounts. ScFv therefore represent valuable molecules for the targeted delivery of drugs. Toxins or radionuclides to a tumor site and has great potential for the development of therapeutic or diagnostic reagents.
One successful approach to recombinant antibody production has been developed ,This approach relies on a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface. Since the displayed antibody retains its antigen-binding capability, it is thus possible to enrich for recombinant phage expressing specific antibodies by affinity selection. With this approach, antibodies of defined specificity and affinity can be selected from a population. Recombinant phage antibody technology has the power and versatility to mimic the features of immune diversity and selection. In time, it may even be possible to replace the current practices of animal immunization and hybridoma development with a bacterial system capable of synthesizing and
expressing virtually unlimited quantities of antibodies to practically any antigen. Phage antibody genes can be easily sequenced, mutated and screened to improve antigen binding. It is even possible to rearrange the genes which code for the various regions of an antibody molecule such that its specificity and affinity for an antigen are altered (3). The aim of the present study was to construct anti-ovarian cancer scFv genes library and obtaine ScFv fragments with binding activities to ovarian cancer antigen By recombinant phage antibody technique,which paves a way for the study of prevention and cure of ovarian cancer.
Since epithelial ovarian carcinoma still remains the most lethal of gynecologic cancers, ovarian cancer cel
自从杂交瘤技术问世以来，单克隆抗体在人类肿瘤诊断和治疗上的应用成为人们关注的焦点。然而，单克隆抗体在肿瘤诊断治疗上所取得的成就并不大，因为抗体的应用有很高的要求。例如，治疗性的或用于体内诊断的抗体片段要求肿瘤穿透力强，在血液中清除更快、免疫原性低等。同时，还需要一个更有效的方法，可以很容易得到对每一个抗原都有优惠特性和有效作用的分子，并达到高水平产量，以满足抗体应用的高标准和日益增加的应用量。由于大多数单克隆抗体是鼠源的，且体积相对较大，故其作为治疗和诊断试剂的主要限制是肿瘤穿透力较差及其免疫原性。此外，直到最近，单克隆抗体的产量依旧受限于涉及到动物免疫和／或杂交瘤制备这一非常耗时耗力的过程。单链抗体片段(scFv)因此成为一种特别流行的抗体形式。该分子的体积减小只有抗原结合部分，由抗体重链可变区(VH)和轻链可变区(VL)经过一个弹性linker连接而成。与完整抗体IgG及抗体Fab段相比，scFv片
第四军医大学博士学位论文 复6页
段相对小的体积和没有k段，使其能更快穿透肿瘤，在血液中更快清除、免
疫原性低，且能很容易的用来构建含有药物或细胞因子的嵌合分子、双价或
双特异抗体。此外鼠s。Fv可通过基因工程的方法修饰他们的亲和性或将鼠的
框架置换为人的来进一步提高他们在人体内的作用，而且大多数ScFv片段
能很容易的在大肠杆菌中重组表达获得足够的量。因此S。FV成为将药物、
毒素、放射性毒素导向肿瘤的很有价值的分子，在治疗和诊断试剂的发展上
具有巨大的潜力。
目前，成功生产重组抗体的方法已研制出来，此方法依赖于将抗体片段
以融合蛋白呈现在噬菌体表面的噬菌体呈现系统。由于呈现的抗体保持着它
的抗原结合活性，因此可通过亲和筛选富集表达特异性抗体的重组噬菌体。
使用这种方法，具有特异性和亲和活性的抗体便能从富集重组噬菌体中筛选
出来。重组噬菌体技术能有效的模仿免疫多样性和选择性的特征，能很容易
的通过测序、突变和筛选来提高重组噬菌体抗体对抗原的结合力，并可用细
菌系统来代替目前的动物免疫和杂交瘤制备，无限量的合成和表达实际上可
针对任何抗原的抗体分子。本研究的目的就是利用噬菌体表面呈现技术构建
一个较大容量的抗人卵巢癌单链抗体基因文库，并表达出活性片段，以期更
早应用于临床，对卵巢癌的诊断、治疗水平的提高有所帮助。
由于上皮性卵巢癌在卵巢癌中致死率中一直最高，因此我们选用来自上
皮性卵巢癌的细胞株HO七，SKOV3，COCI和组织做抗原免疫6七周的
Balb／c小鼠从免疫小鼠脾脏中分离tnRNA，RTPcR分别扩增抗体重、轻链
可变区基因，二者经一个特殊构建的 Linkef（Gly4Sef）连接形成 scFv，然后
克隆到高效噬菌粒载体pCANTABSE中，并在大肠杆菌中表达。经M13KO7
辅助噬菌体挽救后，产生呈现抗体sCFv片段的重组噬菌体。获得库容量约为
4．5 xl 06噬菌体 ScFv库，经过四轮 palllling淘筛后，能与特异性抗原结合的噬
菌体呈现的抗体被筛选出来和富集，噬菌体呈现的重组抗体用ELISA进行检
测和鉴别。结果显示：从随机挑取的 94个噬菌体克隆中筛选到 31个具有抗
DePGrtfllBltt ofobsW and GynecolbglL Xi JwHoWI，m－ FMMU
g
纷四军医大学博士学位论文 第7更
原结合活性的阳性克隆，所有的阳性克隆与其他肿瘤细胞、人正常成纤维细
胞进行ELISA检测，无阳性反应。说明通过pannillg淘筛，从构建的噬菌体
抗体文库中成功去除了能与其他肿瘤细胞和人正常细胞共同结合的噬菌体抗
体组分，获得的阳性克隆具有卵巢癌细胞特异性。
由于可溶性重组抗体能快而经济的生产，是非常有用的免疫试剂，因此
抗原阳性的克隆被分离出来后，我们在大肠杆菌中进行可溶性单链抗体片段
的表达，并用能识别 E－tag sk段的 ntiE Ta gtR以验证。可溶性抗体在大肠杆
菌 TG和 HBZI sl细胞中都能产生，但在 HBZISI细胞中的产量较高，因此
我们用抗原阳性的噬菌体感染 HBZISI细胞，加诱导剂 IPTG诱导可溶性
SCFV蛋白的产生。可溶性抗体可能位于培养上清、细菌周质和细菌内，离心
沉淀细胞，制备不同细胞提取物，用 SDS－PAGE和 nti＊Ang进行 Western blot
检测加以证实可溶性抗体集中的位置。结果显示：表达蛋白为分子量～30KDa
的可溶性蛋白，与预期大小相符，证实有可溶性抗体产生，可溶性抗体主要
集中在细菌内和细菌周质中，但只有细菌周质中细胞内的可溶性抗体具有生
物功能活性。表达的重组可溶性抗体继续用 D。t hi。t、流式细胞仪和免疫荧
光竟争抑制实验检测其与抗原的结合活性。结果显示?
Ovarian carcinoma is the leading cause of death from gynecological cancer. Although advances in treatment with cytoreductive surgery and chemotherapy have improved the survial rate in the last decade, in most patients,the disease is diagnosis at an advanced stage,survial is still poor,with a 5-yr survial rate of only 30%. thus requiring new therapeutic modalities.
Since the advent of hybridoma technology, the potential of murine mAbs for therapeutic and diagnostic application in human cancers has been a focus of interest. However, the therapeutic use of monoclonal antibodies in human cancers has emt with limited success, because there are great demands on such applications of antibody fragments. For example, therapeutic antibody fragments require lower immunogenicity, exhibit more rapid blood clearance and better tumor penetration.The high demand for and the increasing number of applications of antibodies require more efficient methods for their high-level production and the availability of molecules with favorable properties for every antigen. As most of McAb are rodent antibodies with relatively large size, The major limitations of monoclonal antibody conjugates as therapeutic agents have
been inadequate tumor penetration and immunogenicity. Furthermore, until recently, the production of antibodies was limited to very laborious and time-consuming processes involving animal immunization schemes and/or hybridoma generation. Thus single-chain Fv (scFv) fragments became a particularly popular antibody format. The size of these molecules is reduced to the antigen-binding part of the antibody, and they contain the variable domains of the heavy and light chain connected via a flexible linker. Compared to intact IgG or Fab fragments, their relatively small size and absence of Fc-effector domains may allow them to penetrate tumors more rapidly, exhibit more rapid blood clearance and lower immunogenicity and can readily be used to construct chimeric molecules containing drus or cytokines or bivalent and bispecific antibodies, In addition, Murine scFv can be also engineered for improving their performances in vivo in humans by modulating their affinity or by replacing murine by human frameworks and Most scFv fragments can be obtained easily from recombinant expression in Escherichia coll in sufficient amounts. ScFv therefore represent valuable molecules for the targeted delivery of drugs. Toxins or radionuclides to a tumor site and has great potential for the development of therapeutic or diagnostic reagents.
One successful approach to recombinant antibody production has been developed ,This approach relies on a phage-display system in which fragments of antibodies are expressed as fusion proteins displayed on the phage surface. Since the displayed antibody retains its antigen-binding capability, it is thus possible to enrich for recombinant phage expressing specific antibodies by affinity selection. With this approach, antibodies of defined specificity and affinity can be selected from a population. Recombinant phage antibody technology has the power and versatility to mimic the features of immune diversity and selection. In time, it may even be possible to replace the current practices of animal immunization and hybridoma development with a bacterial system capable of synthesizing and
expressing virtually unlimited quantities of antibodies to practically any antigen. Phage antibody genes can be easily sequenced, mutated and screened to improve antigen binding. It is even possible to rearrange the genes which code for the various regions of an antibody molecule such that its specificity and affinity for an antigen are altered (3). The aim of the present study was to construct anti-ovarian cancer scFv genes library and obtaine ScFv fragments with binding activities to ovarian cancer antigen By recombinant phage antibody technique,which paves a way for the study of prevention and cure of ovarian cancer.
Since epithelial ovarian carcinoma still remains the most lethal of gynecologic cancers, ovarian cancer cel
引文
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54. Kabat EA, Wu TT. Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. J Immunol 1991; 147:1709
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56. Kipriyanov SM, Moldenhaner G, Little M et al. High level production of soluble single chain antibodies in small-scale Escherichia coli cultures [J]. J Immunol Metho, 1997;200:69-77
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61. Li, Y, Cockburn W, Kilpatrick JB and Whitelam GC,. High affinity ScFvs from a single rabbit immunized with multiple haptens[J]. Biochem Biophys Res Commun, 2000; 268: 398-404.
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63. Low, NM, Holliger, PH and Winter, G. Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain. J Mol Biol, 1996:260:. 359-368.
64. Luo D, Geng M, Noujaim T et al. An engineered bivalent single-chain antibody fragment that increases antigen binding activity [J]. J Biochem, 1997; 121(5):111-114
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71. Michelsen BK. Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase[J]. Analytical Biochemistry 1995; 225: 172-17
72. Mullaney JM and Black LW. Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure[J]. J Mol Bio, 1 1998; 283: 913-929.
73. Nieba L, Honegger A, Krebber C and Plückthun A. Disrupting the hydro phobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment[J]. Protein Eng, 1997; 10:435-444.
74. Nissim A, Hoogenboom HR., Tomlinson IM, et al. Antibody fragments from a "single pot" phage display library as immunochemical reagents[J]. EMBO, 1994; 13: 692-698.
75. Ohage E andSteipe B. Intrabody construction and expression. I. The critical role of VL domain stability[J]. J. Mol. Biol,. 1999; 291: 1119-1128.
76. Pace CN. easuring and increasing protein stability[J]. Trends Biotechnol. 1990; 8: 93-98.
77. Perez L, Vazquez JE, Ayala M et al. Production, purification and characterization of an (anti-carcinoembryonic antigen) recombinant single-chain Fv antibody fragment [J]. Biotechnol Appl Biochem, 1996; 24(pt1): 79-82
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