人源性噬菌體抗體庫(kù)的構(gòu)建和CEA抗體的篩選及表達(dá)
發(fā)布時(shí)間：2018-12-24 09:40
【摘要】： CEA是人們認(rèn)識(shí)最早的腫瘤相關(guān)抗原之一,它在胎兒的結(jié)腸表面表達(dá)量很高,而在成人CEA濃度很低。但成人患惡性腫瘤和某些炎性疾病時(shí),CEA表達(dá)量會(huì)明顯升高。統(tǒng)計(jì)學(xué)資料表明大腸癌的CEA表達(dá)率為83.3%,肝癌為80%,肺癌為76.6%,乳腺癌為63.2%。CEA本質(zhì)是一類結(jié)構(gòu)復(fù)雜的糖蛋白,分子量180kDa。免疫熒光技術(shù)證明CEA存在于腫瘤細(xì)胞膜上,是細(xì)胞膜的結(jié)構(gòu)蛋白,且在細(xì)胞膜上的表達(dá)率有所不同。雜交瘤技術(shù)的建立使CEA單抗廣泛用于生物學(xué)研究和醫(yī)學(xué)檢測(cè)與腫瘤治療,但其鼠源性引起的人抗鼠反應(yīng)(HAMA)以及完整單抗分子過大難以進(jìn)入腫瘤組織內(nèi)限制了其發(fā)展。噬菌體展示技術(shù)的出現(xiàn)使得在細(xì)菌中克隆人的抗體基因、構(gòu)建抗體文庫(kù)、制備人源單克隆抗體成為現(xiàn)實(shí),尤其通過該技術(shù)在體外模擬抗體生成過程,達(dá)到了不經(jīng)免疫制備抗體,展示了誘人的前景。 在構(gòu)建抗體庫(kù)時(shí)有諸多因素對(duì)庫(kù)的質(zhì)量有影響,最重要的是抗體基因的多樣性和成熟度,以及庫(kù)容量的大小。因此,我們?cè)诮◣?kù)時(shí)注意到(1)標(biāo)本取自腸系膜淋巴結(jié)。淋巴結(jié)是哺乳類特有的,產(chǎn)生免疫應(yīng)答的重要器官。其淋巴小結(jié)內(nèi)95%的細(xì)胞為B細(xì)胞,且大多為轉(zhuǎn)化的大B細(xì)胞。這些B細(xì)胞受濾泡樹突細(xì)胞表面聚集的抗原的選擇作用,已經(jīng)過數(shù)次分裂和膜抗體結(jié)構(gòu)突變過程,只有其膜抗體與表面抗原有高度親和性的細(xì)胞才能保留繼續(xù)分裂和分化,其余的則均被淘汰,所以選擇淋巴結(jié)最終會(huì)獲得高親合力的抗體。(2)標(biāo)本來源于高表達(dá)CEA(10ng/ml)的結(jié)、直腸癌患者,以使得細(xì)胞中含高豐度的抗體mRNA。(3)提取總RNA時(shí),強(qiáng)調(diào)RNA的完整性,增加提取和純化RNA的步驟會(huì)減少RNA的多樣性,影響庫(kù)容,所以RNA的純度達(dá)到OD260/OD280的值在1.6以上即可。(4)先構(gòu)建輕鏈庫(kù),后與克隆的重鏈基因相連,保證重鏈基因的多樣性。(5)制備高質(zhì)量的感受態(tài)細(xì)胞,嚴(yán)格電轉(zhuǎn)化操作,使空質(zhì)粒轉(zhuǎn)化率達(dá)到109才能保證庫(kù)容達(dá)到107以上。從大腸癌患者的腸系膜淋巴結(jié)中一步法提取淋巴細(xì)胞總RNA約20μg,逆轉(zhuǎn)錄合成cDNA,將十個(gè)患者的cDNA混合在一起,用PCR擴(kuò)增κ鏈和Fd段的基因(Fd段約700bp,κ鏈約640bp)。κ鏈基因經(jīng)純化、酶切、連接和電轉(zhuǎn)化,構(gòu)建κ鏈基因文庫(kù),含2.5×107個(gè)轉(zhuǎn)化子,隨機(jī)挑取10個(gè)菌落行SacI+XbaI酶切檢測(cè),重組率為70%。Fd段擴(kuò)增產(chǎn)物克隆到κ鏈基因文庫(kù)質(zhì)粒中,計(jì)數(shù)集落測(cè)定庫(kù)容為5.2×106。XhoI+SpeI酶切檢測(cè),Fd段重組率為90%。用SacI+SpeI酶切檢測(cè),Fab(約1400bp)重組率為30%。 在篩選抗體庫(kù)過程中,噬菌體種類、固相介質(zhì)表面抗原密度或溶液中抗原濃度和清洗時(shí)間三個(gè)因素對(duì)篩選效率影響最大。噬菌體的影響隨種類不同,沒有一定規(guī)律,后兩個(gè)因素是指篩選的嚴(yán)密度。因本次構(gòu)建的抗體庫(kù)Fab重組率為30%,所以采用了五輪篩選,前兩輪采用中等的嚴(yán)密度,避免高親和力但低表達(dá)的噬菌體克隆丟失。后三輪嚴(yán)格篩選條件達(dá)到了篩選目的。第五輪Fab重組率達(dá)到100%,噬菌體滴度比第三輪增加了30倍。從第五輪篩選后得到的細(xì)菌菌落中挑取30個(gè)克隆制備單克隆噬菌體抗體,用ELISA測(cè)定其抗原結(jié)合活性,其中有6個(gè)克隆呈陽(yáng)性(以P/N4判定為陽(yáng)性),陽(yáng)性率為20%。 挑選4株陽(yáng)性噬菌體抗體感染大腸桿菌XL1-Blue,擴(kuò)增后提取噬菌粒,構(gòu)建可溶性Fab表達(dá)載體,轉(zhuǎn)化大腸桿菌XL1-blue,經(jīng)IPTG誘導(dǎo)后收集培養(yǎng)液上清及菌體裂解液上清。ELISA、Western blot檢測(cè)均證實(shí)有3個(gè)克隆成功地表達(dá)了可溶性的Fab抗體蛋白,可溶性抗體與人CEA有特異結(jié)合活性。通過對(duì)克隆1的可變區(qū)進(jìn)行序列測(cè)定分析與GenBank檢索,證實(shí)其與人免疫球蛋白IgG重鏈VH3堿基同源性為90%,氨基酸同源性為88.9%,差異主要集中在CDR2、CDR3區(qū),輕鏈V區(qū)與人免疫球蛋白VK1堿基同源性為93%,氨基酸同源性為91%,CDR1、CDR2、CDR3區(qū)均存在氨基酸差異。根據(jù)同一家族中核苷酸序列同源性大于80%,說明所獲抗體基因重鏈屬于人抗體VH3家族,輕鏈屬于人抗體VK家族。 本研究以噬菌體展示技術(shù)得到了抗CEA的單克隆抗體并證實(shí)了該抗體的有效性,希望使之成為一種理想的免疫治療的藥物,為研究表達(dá)CEA的腫瘤患者的治療提供有益資料。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.11
本文編號(hào)：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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392.11
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前2條
1 閆曉敏;魚源Fab噬菌體抗體庫(kù)的篩選、鑒定及抗SAs抗體的可溶性表達(dá)[D];華中農(nóng)業(yè)大學(xué);2010年
2 王輝;斑點(diǎn)叉尾洶天然單鏈?zhǔn)删w抗體庫(kù)的構(gòu)建及初步鑒定[D];華中農(nóng)業(yè)大學(xué);2011年
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