应用蛋白质组学及噬菌体抗体库技术筛选大肠癌特异性抗原及抗体方法的建立
摘要
寻找大肠癌相关特异性抗原以及高特异性，高亲和性抗体一直备受学者关注。80年代中后期发展起来的噬菌体抗体库技术，使抗体技术进入第三代—基因工程阶段，它具有人源性基因工程抗体异源性弱；克服了杂交瘤技术制备人源性mAb遇到的困难；半衰期延长；能够模拟天然抗体库而无须免疫人和动物；能够模拟体内抗体亲和力成熟过程，获得不同亲和力抗体等优势和特点。构建足够库容的抗体库是筛选目的抗体的基础。90年代发展起来的蛋白质组学及相关技术为有效分离和鉴定蛋白提供了技术平台。噬菌体抗体库技术和蛋白质组学相关技术是目前唯一的在数量，速度及所产生的信息流上能够相互匹配的生物学技术。鉴于蛋白质组学技术具有能够分离，纯化和鉴定特殊蛋白的优势和特点以及现有的筛选肿瘤噬菌体抗体库方法上的不足。我们想到尝试通过Western-Blot免疫印记实验的桥梁，将蛋白质组学相关技术应用到肿瘤抗体库的筛选，这样不仅可以发现和鉴定肿瘤特异性抗原，同时还可以筛选和富集制备与之相应的肿瘤特异性抗体。
材料和方法
1.1 材料
1.1.1 病例选择
随机选取5例于我科行大肠腺癌（C.D期）根治术患者，术中剥取
系膜内转移淋巴结3-5枚，切取大肠癌组织及正常大肠粘膜层组织各1克左右。随即置液氮中保存备用。
1.1.2 载体.菌株和辅助噬菌体
采用噬菌体载体pComb3,由The Scripps Research Institute提供；宿主菌采用大肠杆菌XLI—Blu,由The Scripps Research Institute提供；辅助噬菌体采用VCSM13，由The Scripps Research Institute提供。
1.1.3主要试剂：
RT-PCR和PCR试剂盒购自Promega，限制性内切酶和连接酶购自 Promega.
固相pH梯度干胶条(pH3-10，17cm)、泡胀液，PVDF膜及矿物油均为美国Bi0Rad公司产品。碘乙酞胺、a一晴基一4一羟基肉桂酸、促肾上腺皮质激素片段18一39(ACTH)为5 1 gma公司产品,三氟乙酸(TFA)购自Fluka公司,乙晴(ACN)购自Fisher公司,胰蛋白酶购自Boehringer公司,二硫代苏糖醇(DTT)为Promega公司产品,甘氨酸、Tris、SDS、丙烯酞胺、甲基双丙烯酞胺是上海生物工程公司进口分装产品,低分子量标准蛋白质为上海西巴斯生物技术开发有限公司产品。
1.2方法
1.2.1 转移淋巴结总RNA提取：将新鲜剥取的大肠癌患者肿大淋巴结剖为两半（一半经病理证实为转移淋巴结），剩余部分于液氮中研成粉末状，采用TRIZOL一步法提取组织总RNA。
1.2.2 cDNA的合成：采用RT-PCR试剂盒（Promega公司），方法参照说明书。
1.2.3 PCR扩增重链Fd和轻链基因：引物设计参照The Scripps Research Institute提供的引物组，方法参照PCR试剂盒说明书。
1.2.4 轻链基因和重链Fd基因PCR产物重组入pComb3及噬菌体抗体库的建立：采用Spin-X纯化方法，将轻链基因PCR扩增产物分离纯化，DNA纯化试剂盒制备pComb3载体DNA，以SacI/XbaI双酶切，扩
增后电泳纯化。轻链基因和线性pComb3进行连接，连接产物电转感受态XLI—Blu,扩增培养后，取10ul至50ul培养液涂于含氨苄青霉素的LB平皿，检测转化效率，随机挑取20个单菌落，提取质粒DNA，以SacI/XbaI双酶切，鉴定插入率。采用Spin-X纯化方法将重链Fd基因PCR扩增产物分离纯化，DNA纯化试剂盒制备有轻链基因插入的pComb3载体DNA pComb3-L，以SpeI/XhoI双酶切，扩增后电泳纯化,同法将两者连接并电转感受态XLI—Blu，检测转化效率，以SpeI/XhoI双酶切鉴定轻链基因插入率，以XbaI/XhoI双酶切鉴定同时有轻链基因和重链Fd基因插入的效率。加入辅助噬菌体VCSM13超感染，加PEG和NaCL沉淀，离心，经PBS重悬后，上清即为噬菌体Fab初级抗体库。
1.2.5 免疫印记法鉴定初级抗体库中噬菌体上有Fab段的表达：将Fab初级抗体库悬液直接点于硝酸纤维膜（NC）片上，以pComb3噬菌体空载体悬液作阴性对照，直径0.5厘米，每片5ul。室温自然干燥。以辣根过氧化物酶（HRP）标记的羊抗人IgG为试剂行斑点印记检测。
1.2.6 大肠癌组织和正常大肠粘膜组织匀浆的2—DE电泳及胶图的银染和分析：将5例患者的大肠癌组织和正常大肠粘膜组织分别行研磨.匀浆.超声粉碎后，离心收取上清，加样，分别进行双向电泳，每个样品重复三次，以确保结果的可靠。获得2-DE胶图后，行硝酸银染色。将显色后的凝胶用SONY数码相机照相，保存图象。将每个凝胶多肽模式用Melanie II 2-DE分析软件进行分析。建立大肠癌组织和正常大肠粘膜组织的平均标准胶图。
1.2.7 从2-DE胶到PVDF膜的转移电泳：从每例患者的大肠癌和正常大肠粘膜2-DE胶图中各选取两张质量较好者，进行电转PVDF膜，并分类为：阳性组（大肠癌组）：10张；阴性对照组（正常大肠粘膜组）：10张。
1.2.8 噬菌体Fab抗体库的筛选和富集:将5例患者的大肠癌组织匀浆包被于微孔板，并以其为靶抗原对所建初级抗体库进行淘洗，将每轮
洗脱下来的特异性吸附的噬菌体再感染新鲜的XLI—Blu菌, （感染前取10ul，1ul，0.1ul菌液涂于LB氨苄平皿，以滴定洗脱下来的噬菌体）经辅助噬菌体VCSM13超感染后再行下一轮淘洗，行三轮淘洗后，计算富集率。
1.2.9 Western blot印迹法检测PVDF膜上能与噬菌体抗体结合的抗原：将阳性组和阴性组各10张PVDF膜分?
Looking for the specificity related antigen and the high specificities, high affinity antibody in large intestine carcinoma has been the concern of scholars.Phage antibody library technique that developments in the late 1980's ,make the technique of antibody entered in the 3rd generation- the genetic engineering stage, which has advantage and characteristics envolving weakening antibody heterology with the technique of human source genetic engineering and Overcoming the difficulty that encounters in making human source mAb by hybridization neoplasm technique、extensioning Half-life 、imitating the natural antibody library and need not immune human and animal、imitate the mature process in affinity of antibody inside the body, acquire different affinity antibody etc.To Set up a antibody liberary large enough is the foundation of filteing the aim genes .Proteomics and its related techniques that developments since the 1990's provided the technique terrace of effectively separating and identifying proteins.phage antibody library technique and Proteomics and its related technique is currently the unique biology technique which is mutually matched in quantity, speed and its message flowing.owing to the advantages of the Proteomics technique in separating, purifying and identifying special proteins and the shortcomings of current tumor phage antibody library filtering method .We thought of the method of applying Proteomics and its related techniques to the sieving of the tumor antibody library by the Western- Blot immunity prints trials, which can not only discovers and identify tumor specific antigen
but also sieve and make the homologous tumor specificity antibody
1. Materials and methods
1.1 Materials
1.1.1 Case selection
Randomly selects 5 patients in our department who received the radical surgery of large intestine adenocarcinoma( the period of C.D), shelling 3-5 lymph nodes which had transferred inside the shell membrane, cutting the intestine carcinoma tissue and normal large intestine mucous membrane tissues each 1g or so.Place them immediately to the liquid. nitrogekeep to back up
1.1.2 Carrier、Vaccine and Assistance phage
Adopting bacteriophage carrier-pComb3, offered by The Scripps Research Institute.Host mold adopted escherichia –coli XLI—Blu,offering by The Scripps Research Institute.Assistance bacteriophage adoption VCSM13, offered by The Scripps Research Institute.Assistant phage adopted VCSM13,offered by the Scripps Research Institute.
1.1.3 Major reagent
The RT- PCR and PCR reagent box buy from the Promega, the the restriction endonucleases and ligase buy from the Promega.
The pH gradient trunks , bulge liquid, PVDF membrane and mineral oil are all the products of Bi0Rad company in the United States.The Iodine 、a－nitrile－4－light cinnamon acid、the fragment of corticotropin 18-39( ACTH) are all products of the 51gma company`, three fluoride acetate ( TFA)s buy from the company of Fluka, acenitrile ( ACN) buy from the company of Fisher, the enzyme of trypsin buy from the company of Boehringer, two thiourea on behalf threose ketolase( DTT) for Promega company product、glycine、Tris、SDS、is from the living creature engineering company of Shanghai to import to load separately the product, low molecular standard protein is from a limit company in Shanghai
1.2 Method
1.2.1 The distill of the total RNA in the transferred lymph node:
Dissecting the fresh swollen lymph nodes which were got from the
patient with large intestine cancer into two parts(half of them are proved to be transferred lymph nodes),and then grinding the surplus part into farina in liquid nitrogen to distill the total RNA of tissue through TRIZOL.
1.2.2 Synthesis of the cDNA :
using the RT- PCR ( the company of Promega)
1.2.3 the enlargement of the heavy chain Fd and light chain gene through PCR:
The design of the primer accords to the primer team provided by The Scripps Research Institute ,the method accords to the PCR reagent box manual.
1.2.4 The establish of light chain g
材料和方法
1.1 材料
1.1.1 病例选择
随机选取5例于我科行大肠腺癌（C.D期）根治术患者，术中剥取
系膜内转移淋巴结3-5枚，切取大肠癌组织及正常大肠粘膜层组织各1克左右。随即置液氮中保存备用。
1.1.2 载体.菌株和辅助噬菌体
采用噬菌体载体pComb3,由The Scripps Research Institute提供；宿主菌采用大肠杆菌XLI—Blu,由The Scripps Research Institute提供；辅助噬菌体采用VCSM13，由The Scripps Research Institute提供。
1.1.3主要试剂：
RT-PCR和PCR试剂盒购自Promega，限制性内切酶和连接酶购自 Promega.
固相pH梯度干胶条(pH3-10，17cm)、泡胀液，PVDF膜及矿物油均为美国Bi0Rad公司产品。碘乙酞胺、a一晴基一4一羟基肉桂酸、促肾上腺皮质激素片段18一39(ACTH)为5 1 gma公司产品,三氟乙酸(TFA)购自Fluka公司,乙晴(ACN)购自Fisher公司,胰蛋白酶购自Boehringer公司,二硫代苏糖醇(DTT)为Promega公司产品,甘氨酸、Tris、SDS、丙烯酞胺、甲基双丙烯酞胺是上海生物工程公司进口分装产品,低分子量标准蛋白质为上海西巴斯生物技术开发有限公司产品。
1.2方法
1.2.1 转移淋巴结总RNA提取：将新鲜剥取的大肠癌患者肿大淋巴结剖为两半（一半经病理证实为转移淋巴结），剩余部分于液氮中研成粉末状，采用TRIZOL一步法提取组织总RNA。
1.2.2 cDNA的合成：采用RT-PCR试剂盒（Promega公司），方法参照说明书。
1.2.3 PCR扩增重链Fd和轻链基因：引物设计参照The Scripps Research Institute提供的引物组，方法参照PCR试剂盒说明书。
1.2.4 轻链基因和重链Fd基因PCR产物重组入pComb3及噬菌体抗体库的建立：采用Spin-X纯化方法，将轻链基因PCR扩增产物分离纯化，DNA纯化试剂盒制备pComb3载体DNA，以SacI/XbaI双酶切，扩
增后电泳纯化。轻链基因和线性pComb3进行连接，连接产物电转感受态XLI—Blu,扩增培养后，取10ul至50ul培养液涂于含氨苄青霉素的LB平皿，检测转化效率，随机挑取20个单菌落，提取质粒DNA，以SacI/XbaI双酶切，鉴定插入率。采用Spin-X纯化方法将重链Fd基因PCR扩增产物分离纯化，DNA纯化试剂盒制备有轻链基因插入的pComb3载体DNA pComb3-L，以SpeI/XhoI双酶切，扩增后电泳纯化,同法将两者连接并电转感受态XLI—Blu，检测转化效率，以SpeI/XhoI双酶切鉴定轻链基因插入率，以XbaI/XhoI双酶切鉴定同时有轻链基因和重链Fd基因插入的效率。加入辅助噬菌体VCSM13超感染，加PEG和NaCL沉淀，离心，经PBS重悬后，上清即为噬菌体Fab初级抗体库。
1.2.5 免疫印记法鉴定初级抗体库中噬菌体上有Fab段的表达：将Fab初级抗体库悬液直接点于硝酸纤维膜（NC）片上，以pComb3噬菌体空载体悬液作阴性对照，直径0.5厘米，每片5ul。室温自然干燥。以辣根过氧化物酶（HRP）标记的羊抗人IgG为试剂行斑点印记检测。
1.2.6 大肠癌组织和正常大肠粘膜组织匀浆的2—DE电泳及胶图的银染和分析：将5例患者的大肠癌组织和正常大肠粘膜组织分别行研磨.匀浆.超声粉碎后，离心收取上清，加样，分别进行双向电泳，每个样品重复三次，以确保结果的可靠。获得2-DE胶图后，行硝酸银染色。将显色后的凝胶用SONY数码相机照相，保存图象。将每个凝胶多肽模式用Melanie II 2-DE分析软件进行分析。建立大肠癌组织和正常大肠粘膜组织的平均标准胶图。
1.2.7 从2-DE胶到PVDF膜的转移电泳：从每例患者的大肠癌和正常大肠粘膜2-DE胶图中各选取两张质量较好者，进行电转PVDF膜，并分类为：阳性组（大肠癌组）：10张；阴性对照组（正常大肠粘膜组）：10张。
1.2.8 噬菌体Fab抗体库的筛选和富集:将5例患者的大肠癌组织匀浆包被于微孔板，并以其为靶抗原对所建初级抗体库进行淘洗，将每轮
洗脱下来的特异性吸附的噬菌体再感染新鲜的XLI—Blu菌, （感染前取10ul，1ul，0.1ul菌液涂于LB氨苄平皿，以滴定洗脱下来的噬菌体）经辅助噬菌体VCSM13超感染后再行下一轮淘洗，行三轮淘洗后，计算富集率。
1.2.9 Western blot印迹法检测PVDF膜上能与噬菌体抗体结合的抗原：将阳性组和阴性组各10张PVDF膜分?
Looking for the specificity related antigen and the high specificities, high affinity antibody in large intestine carcinoma has been the concern of scholars.Phage antibody library technique that developments in the late 1980's ,make the technique of antibody entered in the 3rd generation- the genetic engineering stage, which has advantage and characteristics envolving weakening antibody heterology with the technique of human source genetic engineering and Overcoming the difficulty that encounters in making human source mAb by hybridization neoplasm technique、extensioning Half-life 、imitating the natural antibody library and need not immune human and animal、imitate the mature process in affinity of antibody inside the body, acquire different affinity antibody etc.To Set up a antibody liberary large enough is the foundation of filteing the aim genes .Proteomics and its related techniques that developments since the 1990's provided the technique terrace of effectively separating and identifying proteins.phage antibody library technique and Proteomics and its related technique is currently the unique biology technique which is mutually matched in quantity, speed and its message flowing.owing to the advantages of the Proteomics technique in separating, purifying and identifying special proteins and the shortcomings of current tumor phage antibody library filtering method .We thought of the method of applying Proteomics and its related techniques to the sieving of the tumor antibody library by the Western- Blot immunity prints trials, which can not only discovers and identify tumor specific antigen
but also sieve and make the homologous tumor specificity antibody
1. Materials and methods
1.1 Materials
1.1.1 Case selection
Randomly selects 5 patients in our department who received the radical surgery of large intestine adenocarcinoma( the period of C.D), shelling 3-5 lymph nodes which had transferred inside the shell membrane, cutting the intestine carcinoma tissue and normal large intestine mucous membrane tissues each 1g or so.Place them immediately to the liquid. nitrogekeep to back up
1.1.2 Carrier、Vaccine and Assistance phage
Adopting bacteriophage carrier-pComb3, offered by The Scripps Research Institute.Host mold adopted escherichia –coli XLI—Blu,offering by The Scripps Research Institute.Assistance bacteriophage adoption VCSM13, offered by The Scripps Research Institute.Assistant phage adopted VCSM13,offered by the Scripps Research Institute.
1.1.3 Major reagent
The RT- PCR and PCR reagent box buy from the Promega, the the restriction endonucleases and ligase buy from the Promega.
The pH gradient trunks , bulge liquid, PVDF membrane and mineral oil are all the products of Bi0Rad company in the United States.The Iodine 、a－nitrile－4－light cinnamon acid、the fragment of corticotropin 18-39( ACTH) are all products of the 51gma company`, three fluoride acetate ( TFA)s buy from the company of Fluka, acenitrile ( ACN) buy from the company of Fisher, the enzyme of trypsin buy from the company of Boehringer, two thiourea on behalf threose ketolase( DTT) for Promega company product、glycine、Tris、SDS、is from the living creature engineering company of Shanghai to import to load separately the product, low molecular standard protein is from a limit company in Shanghai
1.2 Method
1.2.1 The distill of the total RNA in the transferred lymph node:
Dissecting the fresh swollen lymph nodes which were got from the
patient with large intestine cancer into two parts(half of them are proved to be transferred lymph nodes),and then grinding the surplus part into farina in liquid nitrogen to distill the total RNA of tissue through TRIZOL.
1.2.2 Synthesis of the cDNA :
using the RT- PCR ( the company of Promega)
1.2.3 the enlargement of the heavy chain Fd and light chain gene through PCR:
The design of the primer accords to the primer team provided by The Scripps Research Institute ,the method accords to the PCR reagent box manual.
1.2.4 The establish of light chain g
引文
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