【作者】 杨福辉；

【导师】 方瑾；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2004， 硕士

【摘要】 前言 大肠癌是发病率很高的恶性肿瘤之一，在我国已居第四位，并且死亡率呈逐年上升趋势，探讨有效的诊断和治疗手段是大肠癌研究的重要课题。用单克隆抗体作为载体的大肠癌导向诊断和治疗已经取得了一定进展，但是完整单抗由于分子量较大，致使穿透组织的能力较差，并且绝大多数为鼠源性，对人有较大的免疫源性，因而在肿瘤的显像和治疗中一直未能得到满意的结果。利用基因工程技术改造而获得的小分子抗体，如Fab和scFv等，由于分子量小(Mr25000～30000Da)，可提高穿透组织的能力和血浆清除率，具有良好的诊断价值，但因其只有一个抗原结合位点，其亲和活性明显低于亲本单抗，同时，这种小分子抗体的分子量过小，在体内及靶组织中的滞留时间短，丰度低，因而极大限制了其在肿瘤治疗中的应用。近年来，人们用scFv为构件所构建出来的基因工程双价抗体分子，如minibody，diabody等，因具有双结合位点，使抗体亲和活性大大提高，其分子量适中(55～80kDa)，既保证了抗体在体内组织的穿透能力，又大大改善了scFv在体内清除过快的缺点，在肿瘤的显像和治疗方面显示出良好的前景。 ND-1是宋今丹教授于1983年以人大肠癌细胞系CCL-187为免疫原制备的鼠抗人大肠癌单克隆抗体，已进行的近千例临床病理检测显示，该单抗对高中分化大肠癌组织具有特异结合活性，并优于目前广泛采用的美国商业产品抗CEA单抗。利用ND-1的可变区基因构建的ND-1scFv已经在大肠杆菌中获得功能性表达，实验证实ND-1scFv具有与亲本单抗ND-1相同的结合活性，在荷瘤裸鼠的体内实验中，ND-1scFv亦显示出快速的显像能力和清除速度。然而，和其他scFv类似，ND-1scFv由于分子量小，结合能力单一，在注入裸鼠体内的初期就在肾脏积累并排除，使大部分剂量清除过快，无法在肿瘤部位充分发挥作用，从而限制了其在大肠癌的放免显像和治疗中的作用。 为了提高ND-1scFv在大肠癌的放免显像和治疗中的作用，克服因分子量过小，亲和能力相对较低导致的清除过快的缺点，本研究首次利用基因工程方法构建ND一1的双价单链抗体ND一lsc(Fv):，该抗体是通过柔性短肤，即4个甘氨酸和1个丝氨酸(Gly4Ser)，以共价键将两个scFv片段连接形成，从而使抗体分子在获得双结合能力的同时也增加了分子量，这样既有利于提高抗体的亲和活性又能改善其在体内清除过快的缺点。ND一lsc(Fv)2的构建为进一步的体内荷瘤裸鼠实验及大肠癌临床放免显像和治疗提供了实验依据。实验材料与方法 利用ND一lseFv表达质粒pET一sa(+)ND一lseFv为模板，通过PCR扩增scFv基因，在扩增过程中通过引物Pl、P2引人编码Gly4Ser的Unker序列及用于将其插人到表达载体pET一8a(+)ND一1 scFv的scFv基因下游所须的酶切位点Sall和凡ndm，PCR产物连接克隆载体pMD18一T形成质粒pMD18一T unker一ND一1 scFv并转化大肠杆菌，测序鉴定单菌落。从经鉴定的单菌落提取质粒，将提取的质粒与pET一sa(+)ND一1 scFv经s公FHindln双酶切、连接形成由编码Gly4 Ser的Unker序列连接的两段scFv基因的串联序列，即pET一sa(+)ND一1 se(Fv):，将其转化大肠杆菌BUI，调整lpTG浓度、时间及温度等诱导条件，通过SDS一PAGE确定最佳诱导条件，以最佳诱导条件大量诱导表达ND一lsc(Fv):，菌体裂解收集包涵体，经变性溶解，Ni-NTA resin亲和层吸柱纯化，复性，纯化后的ND一 lsc(Fv):进行SDS一PAGE电泳测定其分子量及纯度分析，间接免疫荧光显微法检测其与培养中人大肠癌细胞系CCL一187细胞的亲和活性。非竞争EUSA法检测并比较ND-lseFv，ND一lse(Fv):及ND一IM汕之间的亲和活性。实验结果 利用ND一lseFv表达质粒pET一sa(+)ND一lseFv为模板，以引物PI、P2进行PCR，产物经琼脂糖电泳显示为750bP左右，与scFv基因片段大小相符。该产物连接克隆载体pMD18一T形成质粒pMD18.”山众er一ND一lscFv，该质粒经S欲FHindm双酶切鉴定获得75Obp左右产物，与血ker-ND一1 scFv序列大小相符，经DNA测序证明所插人序列Unker一ND一1 scFv与设计序列相符。将质粒pMD18一几nke卜ND一1 scFv进行SalF凡ndln双酶切，产物片段hnker一ND一1 seFv与经SalFHindlll双酶切消化的pET一sa(+)ND一1 seFv连接，转化感受态E.。011 BL21，挑单菌落提质粒，以凡nd HFEcoRI双酶切获得巧oobp左右片段，则质粒即为构建成功的表达质粒pET一sa(+)ND-lsc(Fv):，将该菌落经IPTG诱导表达，SDS一PAGE分析得37℃、1刊毛0.smM、3小时可作为最佳诱导条件。以最佳诱导条件诱导表达后获得包涵体，再经过变性，His·T嗯亲合层析纯化，SDS一PAGE显示蛋白纯度约90%，分子量与预测分子量55kDa相符。蛋白经复性后对大肠癌细胞系CCL一187免疫荧光显示出相对于HeLa细胞明显高的特异性，EUSA检测显示ND一1 se(Fv):具有较ND一1 seFv显著高的亲和活性(p<0.01)。讨论 本研究通过PCR在ND一lscFv基因上游引人用于连接两段ND一lscFv串联基因序列的Unker序列，和基因拼接所须的限制点，通过与ND一lscFv表达载体ND一lscFv基因下游相应限制点双酶切拼接?更多还原

【Abstract】 Colorectal carcinoma is one of the common malignant tumors which have higher incidence, occupying the fourth place in our country and the death rate increasing continuously in recent years. Therefore, efficient diagnosis and therapeutic approaches are important for colorectal carcinoma research. Although in recent years some progress has been made in respect to application of monoclonal antibodies for the therapy and diagnosis of colorectal carcinoma, most mAbs are of murine origin, so that they usually have antigenic activity to human , and their large sizes also lead to poor diffusion and long persistence in circulation, which severely limit the efficacy of clinical utilization. Gene engineered antibody fragments like Fab and scFv which have small molecular weight (Mr,25000 ~30000Da ) can improve diffusion and circulation clearance,while they are low avidity because of their monovalent, and their small sizes lead to too quick clearance in circulation. Recently bivalent antibody fragments which are constructed by using monovalent antibody fragment like scFv and Fab display optimized effects in tumor imaging and therapy, bivalent can promise higher a-vidity than monovalent, and the medial molecular size can guarantee the diffusion and moderate clearance in circulation.ND-lmAb is a murine monoclonal antibody against tumor-associated antigen LEA which mainly expressed in human colorectal carcinoma. It was developed by Professor Song Jindan in 1983 by immunizing Balb/c mice with CCL-187 human colorectal carcinoma cell line. The histological determination of nearly one thousand pathologic samples showed that ND-1 can bind specially to well differentiated and moderately differentiated colorectal carcinoma tissues andits specificity is superior to IgG against CEA which has been commercialized in the US. Single chain Fv of ND-1 (ND-lscFv) against human colorectal carcinoma was constructed by fusing gene of variable fragment of heavy chain and light chain and expressed in E. coli. It showed that ND-lscFv displayed fast imaging and clearance, but for the other part, monovalent and so small size lead to low avidity and low accumulation in tumor tissues, so that limit its function in tumor therapy.Therefore, we constructed the bivalent single chain Fv of ND-1 (ND-lsc (Fv)2) by genetic recombination of the gene of ND-lscFv. It was constructed by a flexible short peptide of 4 gly sines and 1 serine ( Gly4Ser) covalently connected the two scFv antibody fragments. The binding sites and molecular weight have been doubled at the same time, so as to gain high avidity and to overcome shortage of too fast clearance rate of scFv. The construction of ND-lsc( Fv)2 can provide evidence for nude mice bearing experiments and clinical diagnosis and therapy of colorectal carcinoma.Materials and MethodsFrom the expression vector pET-28a( + )ND-lscFv, the gene of ND-lscFv was amplified and added a linker sequence ( encoding Gly4Ser) to the upstream of the gene of ND-lscFv and restrict sites Sail and Hindlll by primer PI and P2. The PCR product was ligated into clone vector of pMD18-T to form pMD18-TLinker-ND-1 scFv. correct clone containing pMDlS-TLinker-ND-lscFv was i-dentified by DNA sequencing. pMD18-TLinker-scFv was double digested by Sail and Hindlll, the product of Linker-scFv was recovered and linked into pET-28a ( + ) ND-1 scFv which is prepared by double digestion by Sail and Hindlll. The novel vector pET-28a( + ) ND-lsc(Fv)2 was transformed into E. coli BL21. Condition for optimal expression was adjusted. To Express ND-lsc(Fv)2 by the optimal condition. Target protein was purified by Ni-NTA resin affinity chroma-tography, and then was refolded, its molecular weight and purity were analyzed by SDS-PAGE. Immunofluorescence Assay was conduct to determinate the specificity of ND-lsc( Fv)2to CCL-187 cell line by comparing to HeLa cell line.The avidity among ND-lmAb ND-lsc ( Fv)2and ND-lscFv was compared by ELISA.ResultsUsing the expression vector pET-28a( + )ND-lscFv as template, PCR produced a about 750bp更多还原