大肠癌噬菌体抗体库的构建及抗CEA抗体的筛选解析

1、大肠癌噬菌体抗体库的构建及抗 CEA抗体的筛选作者：南清振，高蕾，张振书，杨希山，肖冰，姜【摘要】 目的 构建及初步鉴定大肠癌噬菌体抗体 Fab 呈现库，筛选人 CEA 单克隆抗体，并进行序列分析。方法 分离大肠癌患者外周血淋巴细胞，提取淋 巴细胞总RNA逆转录成cDNA用PCRT增人全套抗体基因片段，克隆于 pComb3载体，再经电穿孔转化大肠杆菌 XL1 Blue菌株，形成噬菌体抗体 库。以固相化CEA抗原淘筛抗体库，ELISA鉴定噬菌体抗体。其中一个阳性克 隆进行测序。结果 逆转录PCF分别扩增出约680bp大小的k、入和Fd基因。 PCF产物和载体经纯化、双酶切后进行连接转化，成功地构

2、建了人源性Fab抗体基因库，库容量达2.1 X 107，Fab基因重组率为50%以单抗捕获的CEA抗 原淘洗4轮，出现特异性富集，阳性克隆经直接 ELISA和交叉反应ELISA实验 证实具有良好的抗CEA抗原特异性。DNA测序表明该抗体重链属IgG亚类并含 有一条IgL亚类的轻链。结论 成功构建了大肠癌患者自然致敏抗体 Fab段噬菌 体呈现库，从中获得可与CEA抗原结合的噬菌体抗体，由此为大肠癌早期诊断 及基因治疗提供了一种新的思路和方法。【关键词】大肠肿瘤；噬菌体抗体库；Fab抗体；癌胚抗原Abstract ： Objective To construct the Fab phage dis

3、play antibody library of colorectal cancer so as to screen carcinoembryonic antigen (CEA) phage antibody from the constructed phage display library.MethodsThe total cell RNA was extracted froma colorectal cancer patients peripheral lymphocytes, and reverse transcribed into cDNA. These cDNA were ampl

4、ified to the light and the heavy chain DNA by PCR using relevant primers, and then cloned into the expression pComb3 vector. Through transformed into XL1BlueEscherichia coli by electroporation, the phage display antibody library was constructed successively. After that, CEA antigen was coated to pan

5、 the constructed library in order to select antigen binders, and the selected phage antibodies were assayed by ELISA analysis. One positive clone was analyzed by DNA sequenced.Results The amplified fragments of k，入 and Fd DNA were about 680bp. The amplification products were cloned into the expressi

6、on pComb3 vector and were expressed in XL1Blue Escherichia coli. Ahuman Fab antibody library was constructed with a size of 2.1X107and an efficacy of about 50% ( k，入 and Fd DNA were all inserted). Using coated CEA antigen to pan the phage display antibody library four rounds, the phage antibodies sh

7、owed enrichment specifically. The positive clones had good antiCEA specificity which verified byELISA directly and crossreaction. Through DNA sequencing of onepositive clone, it showed that its heavy chain belonged to IgG subvariety and its light chain to IgL subvariety. ConclusionWesucceed in const

8、ructing a Fab phage display antibodies library with natural immunity by a colorectal cancer patient. From it, we also succeed in obtaining the antibody which has the binding activity to CEA antigen.Key words ： colorectal cancer; phage antibody library; Fab antibody; carcinoembryonic antigen噬菌体抗体库技术将

9、抗体轻链基因和重链基因在体外重组并借助噬菌体表 面展示 （ phage display ）系统对目的抗体基因进行筛选 1 。 该技术能够绕 开细胞杂交瘤技术， 直接从基因水平制备特异性单链抗体，由此开创了简便、 快速生产基因工程抗体的先河，这被认为是继杂交瘤技术后抗体生产技术的又 一次革命。自该技术的问世，已被广泛用于生物医学的许多领域。我们采用噬菌体显示技术构建了人大肠癌自然致敏抗体 Fab 段噬菌体呈现 库，以人CEA乍为目标抗原，制备了人源性的抗 CEA单克隆抗体。1 材料和方法1.1 材料1.1.1 噬粒、菌株、细胞及主要试剂表达噬粒载体pComb3大肠埃希菌菌株 XL1 Blue 和

10、辅助噬菌体 VCSM 13为本研究室保存。淋巴细胞分离液 （天津血液研究所），总RNA提取试剂盒（invitrogen 产品），逆转录试剂盒 （Promega公司）、Taq酶及PCF缓冲液（TaKaRa公司产品），DNA凝胶纯化回收试 剂盒（TaKaRa公司产品），各种限制性内切酶（Promega产品）。羊抗人IgG（Fab）HRP购自Pierce公司，邻苯二胺（OPD购自博士德公司。LB、SBSOC培养基配置参照文献1。1.1.2 PCR引物引物设计参见文献2。由上海生物工程有限公司合成。序列如下：Human variable and constant region PCR primers

11、used for phage library constructionHuman chain variable region 5primers ：VH1a 5 CAG GTG CAG CTC GAG CAG TCT GG3VH1f 5 CAG GTG CAG CTG CTC GAGTCT GGG 3VH2f 5 CAG GTG CAG CTA CTC GAGTCG GG 3VH3a 5 GAG GTG CAG CTC GAG GAG TCT GG3GVH3f 5 GAG GTG CAG CTG CTC GAG TCT GG3GVH4f 5 CAG GTG CAG CTG CTC GAG TCG

12、 G3GVH4gs 5 CAG GTG CAG CTA CTC GAG TGG GG3CVH6a 5 CAG GTA CAG CTC GAG CAG TCA GG3VH6f 5 CAG GTA CAG CTG CTC GAG TCA GGT CC3AHeavy chain 丫 1 constant region 3 primer :CGlz 5GCA TGT ACT AGT TTT GTC ACA AGA TTT GGG3k light chain variable region 5 primes:VK1a 5GAC ATC GAG CTC ACC CAG TCT CCA3VK1s 5GAC

13、ATC GAG CTC ACC CAG TCT CC3VK2a 5GAT ATT GAG CTC ACT CAG TCT CCA3VK3a 5GAA ATT GAG CTC ACG CAG TCT CCA3VK3b 5GAA ATT GAG CTC ACA CAG TCT CCA3k light chain constant region 3 prime :CK1d 5GCG CCG TCT AGA ATT AAC ACT CTC CCC TGT GA AGC TCT TTGTGA CGG GCG AAC TCA G3入 light chain variable region 5 primer

14、s :VL1 5AAT TTT GAG CTC ACT CAG CCC CAC 3VL2 5TCT GCC GAG CTC CAG CCT GCC TCC GTG 3VL3 5TCT GTG GAG CTC CAG CCG CCC TCA GTG3VL3 5 TCC TAT GAG CTC ACT CAG CCA CCC 3VL4 5TCT GAA GAG CTC CAG GAC CCT GTT GTG TCT GTG3VL5 5CAG TCT GAG CTC ACG CAG CCG CCC 3VL6 5 CAG ACT GAG CTC ACT CAG GAG CCC 3VL7 5 CAG GTT GAG CTC ACT CAA CCG CCC 3VL8 5 CAG GCT GAG CTCACT CAG CCG TCT TCC 3VL9 5 CAG TAT GAG CTC ACT CAG CCT CCC 3VL1b/s 5 CAG TCT GAG CTC ACT CAG CCA CC3入 light chain constant region 3 primers :CL2 5 CG CCG TCT AGA ATT ATG AAC ATT CTG TAG G3Underline