人源性噬菌体抗体库的构建和CEA抗体的筛选及表达
发布时间：2018-12-24 09:40
【摘要】： CEA是人们认识最早的肿瘤相关抗原之一,它在胎儿的结肠表面表达量很高,而在成人CEA浓度很低。但成人患恶性肿瘤和某些炎性疾病时,CEA表达量会明显升高。统计学资料表明大肠癌的CEA表达率为83.3%,肝癌为80%,肺癌为76.6%,乳腺癌为63.2%。CEA本质是一类结构复杂的糖蛋白,分子量180kDa。免疫荧光技术证明CEA存在于肿瘤细胞膜上,是细胞膜的结构蛋白,且在细胞膜上的表达率有所不同。杂交瘤技术的建立使CEA单抗广泛用于生物学研究和医学检测与肿瘤治疗,但其鼠源性引起的人抗鼠反应(HAMA)以及完整单抗分子过大难以进入肿瘤组织内限制了其发展。噬菌体展示技术的出现使得在细菌中克隆人的抗体基因、构建抗体文库、制备人源单克隆抗体成为现实,尤其通过该技术在体外模拟抗体生成过程,达到了不经免疫制备抗体,展示了诱人的前景。 在构建抗体库时有诸多因素对库的质量有影响,最重要的是抗体基因的多样性和成熟度,以及库容量的大小。因此,我们在建库时注意到(1)标本取自肠系膜淋巴结。淋巴结是哺乳类特有的,产生免疫应答的重要器官。其淋巴小结内95%的细胞为B细胞,且大多为转化的大B细胞。这些B细胞受滤泡树突细胞表面聚集的抗原的选择作用,已经过数次分裂和膜抗体结构突变过程,只有其膜抗体与表面抗原有高度亲和性的细胞才能保留继续分裂和分化,其余的则均被淘汰,所以选择淋巴结最终会获得高亲合力的抗体。(2)标本来源于高表达CEA(10ng/ml)的结、直肠癌患者,以使得细胞中含高丰度的抗体mRNA。(3)提取总RNA时,强调RNA的完整性,增加提取和纯化RNA的步骤会减少RNA的多样性,影响库容,所以RNA的纯度达到OD260/OD280的值在1.6以上即可。(4)先构建轻链库,后与克隆的重链基因相连,保证重链基因的多样性。(5)制备高质量的感受态细胞,严格电转化操作,使空质粒转化率达到109才能保证库容达到107以上。从大肠癌患者的肠系膜淋巴结中一步法提取淋巴细胞总RNA约20μg,逆转录合成cDNA,将十个患者的cDNA混合在一起,用PCR扩增κ链和Fd段的基因(Fd段约700bp,κ链约640bp)。κ链基因经纯化、酶切、连接和电转化,构建κ链基因文库,含2.5×107个转化子,随机挑取10个菌落行SacI+XbaI酶切检测,重组率为70%。Fd段扩增产物克隆到κ链基因文库质粒中,计数集落测定库容为5.2×106。XhoI+SpeI酶切检测,Fd段重组率为90%。用SacI+SpeI酶切检测,Fab(约1400bp)重组率为30%。 在筛选抗体库过程中,噬菌体种类、固相介质表面抗原密度或溶液中抗原浓度和清洗时间三个因素对筛选效率影响最大。噬菌体的影响随种类不同,没有一定规律,后两个因素是指筛选的严密度。因本次构建的抗体库Fab重组率为30%,所以采用了五轮筛选,前两轮采用中等的严密度,避免高亲和力但低表达的噬菌体克隆丢失。后三轮严格筛选条件达到了筛选目的。第五轮Fab重组率达到100%,噬菌体滴度比第三轮增加了30倍。从第五轮筛选后得到的细菌菌落中挑取30个克隆制备单克隆噬菌体抗体,用ELISA测定其抗原结合活性,其中有6个克隆呈阳性(以P/N4判定为阳性),阳性率为20%。 挑选4株阳性噬菌体抗体感染大肠杆菌XL1-Blue,扩增后提取噬菌粒,构建可溶性Fab表达载体,转化大肠杆菌XL1-blue,经IPTG诱导后收集培养液上清及菌体裂解液上清。ELISA、Western blot检测均证实有3个克隆成功地表达了可溶性的Fab抗体蛋白,可溶性抗体与人CEA有特异结合活性。通过对克隆1的可变区进行序列测定分析与GenBank检索,证实其与人免疫球蛋白IgG重链VH3碱基同源性为90%,氨基酸同源性为88.9%,差异主要集中在CDR2、CDR3区,轻链V区与人免疫球蛋白VK1碱基同源性为93%,氨基酸同源性为91%,CDR1、CDR2、CDR3区均存在氨基酸差异。根据同一家族中核苷酸序列同源性大于80%,说明所获抗体基因重链属于人抗体VH3家族,轻链属于人抗体VK家族。 本研究以噬菌体展示技术得到了抗CEA的单克隆抗体并证实了该抗体的有效性,希望使之成为一种理想的免疫治疗的药物,为研究表达CEA的肿瘤患者的治疗提供有益资料。
[Abstract]:CEA is one of the earliest known tumor-related antigens, which is highly expressed in the colon surface of the fetus and is low in the adult CEA concentration. In adults with malignant and some inflammatory diseases, the level of CEA expression is significantly increased. The statistical data showed that the expression rate of CEA in colorectal cancer was 83.3%, liver cancer was 80%, lung cancer was 76.6%, and breast cancer was 63.2%. The essence of CEA was a kind of glycoprotein with complex structure and molecular weight of 180kDa. Immunofluorescence technique has shown that CEA is present on the cell membrane of the tumor, it is the structural protein of the cell membrane, and the expression rate on the cell membrane is different. The establishment of the hybridoma technique allows CEA monoclonal antibody to be widely used for biological research and medical detection and tumor treatment, but the human anti-mouse reaction (HAMA) and the complete monoclonal antibody of the human-induced human anti-mouse reaction (HAMA) and the complete monoclonal antibody are difficult to enter the tumor tissue to limit the development thereof. The emergence of the phage display technology makes it possible to clone the antibody gene in the bacteria, construct the antibody library, and make the human source monoclonal antibody to be a reality, in particular, the antibody generation process is simulated in vitro by the technology, so that the antibody is not prepared through immunization, and the attractive prospect is displayed. In building the antibody library, there are many factors that affect the quality of the library, and the most important is the diversity and the maturity of the antibody gene. and the size of the library capacity. Therefore, we take note of (1) when we build the library The present invention is derived from the mesenteric lymph node. The lymph node is specific to the mammal and is produced. An important organ of an immune response. 95% of the cells in the lymphoid nodule are B cells and are large A large number of transformed large B cells. These B cells are selected by the antigen selected from the surface of the follicular dendritic cells, and have been subjected to a number of splitting and membrane antibody structure mutation processes. Only cells with high affinity for their membrane antibodies and surface antigens can be retained for continued division and differentiation and the rest is eliminated, so the selection of the lymph node will eventually High-affinity antibodies were obtained. (2) The specimens were derived from high-expression CEA (10ng/ ml) colorectal cancer patients, so that the cells contained high Abundance of the antibody mRNA. (3) When the total RNA is extracted, the integrity of the RNA is emphasized, the steps of increasing the extraction and purification of the RNA can reduce the diversity of the RNA and affect the storage capacity, so that the purity of the RNA reaches the OD260/ OD280. and (4) constructing a light chain library, and then connecting the light chain library with the cloned heavy chain gene, the diversity of the heavy chain gene is proved. (5) high-quality competent cells are prepared, and the transformation operation is carried out strictly, so that the conversion rate of the empty plasmid reaches 109 the total RNA of the lymphocytes was extracted from the mesenteric lymph nodes of the patients with large intestine cancer by about 20. m u.g, the cDNA was synthesized by reverse transcription, and the cDNA of ten patients was mixed together, and the gene (Fd) of the whole chain and the Fd section was amplified by PCR (about 700bp, and carrying out purification, enzyme-cutting, connection and electric transformation of the chain-chain gene, constructing a chain-chain gene library, containing 2-5-107 transformants, randomly selecting 10 colony rows of SacI + XbaI enzyme-cutting detection, and the recombination rate is 70%. in that plasmid of the chain gene library, the capacity of the counting colony was 51.2-106. XhoI + SpeI was cut and tested, F d-segment recombination rate of 90%. Detection with SacI + SpeI, Fab (approximately 14000b p) The recombination rate was 30%. During the screening of the antibody library, the phage species, the solid phase medium surface antigen density or the antigen concentration in the solution The influence of three factors on the screening efficiency is the most important to the screening efficiency. The influence of the phage is different with the species. There is a certain law, the latter two factors are the strict density of screening. The Fab recombination rate of the antibody library constructed in this time is 30%, so the five-wheel screening is adopted, and the first two-wheel adopts the middle strict density. Avoid high-affinity, but low-expressed, phage clones Missing. Three-wheel critical screening conditions were selected for screening purposes. The fifth wheel Fab recombination rate reached 1 30 clones of the bacterial colonies obtained from the fifth wheel were selected to prepare the monoclonal phage antibody, and the antigen binding activity was determined by ELISA, of which 6 clones were positive (The positive rate was 20% (P/ N4). Four positive phage antibodies were selected to infect E. coli XL1-Blue. After amplification, the phagocyte was extracted, and the soluble Fab expression vector was constructed and transformed into E. coli XL1.-blue, after the induction of IPTG, the supernatant was collected and the cell lysate was purified. The ELISA and Western blot showed that 3 clones were successfully expressed. The soluble Fab antibody protein and the soluble antibody have specific binding activity with the human CEA. By sequencing and analyzing the variable region of the clone 1 and the GenBank search, it is confirmed that the homology with the human immunoglobulin IgG heavy chain VH3 base is 90%, the homology of the amino acid is 80.9%, the difference is mainly concentrated on the CDR2, In the CDR3 region, the homology of the light chain V region to the human immunoglobulin VK1 base is 93%, and the amino acid is homologous. The amino acid difference of CDR1, CDR2 and CDR3 region is 91%, and the homology of the nucleotide sequence homology of the same family is more than 80%. The heavy chain of the obtained antibody belongs to the human antibody VH3 family, and the light chain belongs to the human antibody VK family.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.11
本文编号：2390473
[Abstract]:CEA is one of the earliest known tumor-related antigens, which is highly expressed in the colon surface of the fetus and is low in the adult CEA concentration. In adults with malignant and some inflammatory diseases, the level of CEA expression is significantly increased. The statistical data showed that the expression rate of CEA in colorectal cancer was 83.3%, liver cancer was 80%, lung cancer was 76.6%, and breast cancer was 63.2%. The essence of CEA was a kind of glycoprotein with complex structure and molecular weight of 180kDa. Immunofluorescence technique has shown that CEA is present on the cell membrane of the tumor, it is the structural protein of the cell membrane, and the expression rate on the cell membrane is different. The establishment of the hybridoma technique allows CEA monoclonal antibody to be widely used for biological research and medical detection and tumor treatment, but the human anti-mouse reaction (HAMA) and the complete monoclonal antibody of the human-induced human anti-mouse reaction (HAMA) and the complete monoclonal antibody are difficult to enter the tumor tissue to limit the development thereof. The emergence of the phage display technology makes it possible to clone the antibody gene in the bacteria, construct the antibody library, and make the human source monoclonal antibody to be a reality, in particular, the antibody generation process is simulated in vitro by the technology, so that the antibody is not prepared through immunization, and the attractive prospect is displayed. In building the antibody library, there are many factors that affect the quality of the library, and the most important is the diversity and the maturity of the antibody gene. and the size of the library capacity. Therefore, we take note of (1) when we build the library The present invention is derived from the mesenteric lymph node. The lymph node is specific to the mammal and is produced. An important organ of an immune response. 95% of the cells in the lymphoid nodule are B cells and are large A large number of transformed large B cells. These B cells are selected by the antigen selected from the surface of the follicular dendritic cells, and have been subjected to a number of splitting and membrane antibody structure mutation processes. Only cells with high affinity for their membrane antibodies and surface antigens can be retained for continued division and differentiation and the rest is eliminated, so the selection of the lymph node will eventually High-affinity antibodies were obtained. (2) The specimens were derived from high-expression CEA (10ng/ ml) colorectal cancer patients, so that the cells contained high Abundance of the antibody mRNA. (3) When the total RNA is extracted, the integrity of the RNA is emphasized, the steps of increasing the extraction and purification of the RNA can reduce the diversity of the RNA and affect the storage capacity, so that the purity of the RNA reaches the OD260/ OD280. and (4) constructing a light chain library, and then connecting the light chain library with the cloned heavy chain gene, the diversity of the heavy chain gene is proved. (5) high-quality competent cells are prepared, and the transformation operation is carried out strictly, so that the conversion rate of the empty plasmid reaches 109 the total RNA of the lymphocytes was extracted from the mesenteric lymph nodes of the patients with large intestine cancer by about 20. m u.g, the cDNA was synthesized by reverse transcription, and the cDNA of ten patients was mixed together, and the gene (Fd) of the whole chain and the Fd section was amplified by PCR (about 700bp, and carrying out purification, enzyme-cutting, connection and electric transformation of the chain-chain gene, constructing a chain-chain gene library, containing 2-5-107 transformants, randomly selecting 10 colony rows of SacI + XbaI enzyme-cutting detection, and the recombination rate is 70%. in that plasmid of the chain gene library, the capacity of the counting colony was 51.2-106. XhoI + SpeI was cut and tested, F d-segment recombination rate of 90%. Detection with SacI + SpeI, Fab (approximately 14000b p) The recombination rate was 30%. During the screening of the antibody library, the phage species, the solid phase medium surface antigen density or the antigen concentration in the solution The influence of three factors on the screening efficiency is the most important to the screening efficiency. The influence of the phage is different with the species. There is a certain law, the latter two factors are the strict density of screening. The Fab recombination rate of the antibody library constructed in this time is 30%, so the five-wheel screening is adopted, and the first two-wheel adopts the middle strict density. Avoid high-affinity, but low-expressed, phage clones Missing. Three-wheel critical screening conditions were selected for screening purposes. The fifth wheel Fab recombination rate reached 1 30 clones of the bacterial colonies obtained from the fifth wheel were selected to prepare the monoclonal phage antibody, and the antigen binding activity was determined by ELISA, of which 6 clones were positive (The positive rate was 20% (P/ N4). Four positive phage antibodies were selected to infect E. coli XL1-Blue. After amplification, the phagocyte was extracted, and the soluble Fab expression vector was constructed and transformed into E. coli XL1.-blue, after the induction of IPTG, the supernatant was collected and the cell lysate was purified. The ELISA and Western blot showed that 3 clones were successfully expressed. The soluble Fab antibody protein and the soluble antibody have specific binding activity with the human CEA. By sequencing and analyzing the variable region of the clone 1 and the GenBank search, it is confirmed that the homology with the human immunoglobulin IgG heavy chain VH3 base is 90%, the homology of the amino acid is 80.9%, the difference is mainly concentrated on the CDR2, In the CDR3 region, the homology of the light chain V region to the human immunoglobulin VK1 base is 93%, and the amino acid is homologous. The amino acid difference of CDR1, CDR2 and CDR3 region is 91%, and the homology of the nucleotide sequence homology of the same family is more than 80%. The heavy chain of the obtained antibody belongs to the human antibody VH3 family, and the light chain belongs to the human antibody VK family.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.11
【引证文献】
相关硕士学位论文 前2条
1 闫晓敏;鱼源Fab噬菌体抗体库的筛选、鉴定及抗SAs抗体的可溶性表达[D];华中农业大学;2010年
2 王辉;斑点叉尾洶天然单链噬菌体抗体库的构建及初步鉴定[D];华中农业大学;2011年
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