利用噬菌体表面展示技术制备人源性抗肿瘤坏死因子-αFab’抗体
发布时间：2018-08-08 18:26
【摘要】：肿瘤坏死因子-α（TNF-α）是在类风湿性关节炎（RA）等疾病的病理变化中起关键作用的细胞因子，临床试验结果证实，TNF-α是治疗该类疾病的合适靶点。针对TNF-α的抑制剂用于治疗RA取得了良好的效果，其代表药物有Enbrel，Remicade，Humira等。随着TNF抗体类药物临床试验的开展，不断有新的适应症出现，其市场空间不断扩大。这些药物具有特异性高，靶向性好，副作用低的优点，已逐渐成为与氨甲碟呤一起治疗RA的一线用药。但是它们也存在着不同的缺陷，主要有如下两点：第一，Remicade是人鼠嵌合抗体，含有25%的鼠源片段，长期注射可能会引起人抗鼠抗体（HAMA）反应；第二，这些抗体所携带的Fc段具有激活补体和ADCC等活性，会导致表达TNF的细胞凋亡，，产生一系列副作用。因此研发全人源小分子TNF-α抗体具有特别重要的意义。 本课题中全人源小分子TNF-α抗体的研发包括三个部分：TNF-α抗原的表达纯化，抗体库筛选和抗TNF-α Fab’抗体的表达纯化。 TNF-α抗原的表达纯化：根据GenBank中的人源TNF-α序列，全化学合成TNF-α基因，提取基因构建TNF-α表达载体pET-23b/TNF-α并将其转化大肠杆菌BL21(DE3)，获得阳性克隆。将阳性克隆于37℃诱导表达16h得到发酵液上清中可溶性分泌表达的TNF-α，将其浓缩后再通过层析纯化，最终得到了TNF-α纯品，纯度大于95%，比活性为4.4×10~6U/mg，产量为4mg/L。 抗体库筛选：我们利用TNF-α作为抗原对噬菌体抗体库进行筛选，以期获得对TNF-α有特异性亲和力的抗体克隆。经过六轮固相淘洗，与TNF-α特异性结合的噬菌体抗体克隆从1.1×10~3pfu上升至2×10~5pfu，产出投入比从3.2×10~(-8)上升至1.2×10~(-6)，尽管随着筛选轮数的增加洗脱条件不断严格，最终还是得到了200倍的富集。从第六轮淘洗获得的克隆中随机挑选200个克隆进行ELISA筛选，得到5株阳性克隆。进一步对这5株克隆进行抗原结合特异性比较，最终确定了一株与TNF-α特异性最好的克隆G11。 抗TNF-α Fab’抗体的表达纯化：将筛选得到的G11克隆进行测序，获得抗体基因序列，构建Fab’抗体表达载体。将载体转化大肠杆菌BL21(DE3)，获得阳性克隆后，我们对其表达条件进行优化，比较了培养基和诱导时间对表达量的影响，结果显示，用LB培养基在30℃，IPTG诱导浓度0.1mM下，诱导24h，Fab’抗体表达量最高。我们还对细菌破壁方法进行了比较，在超声，渗透压休克、溶菌酶和反复冻融中我们选择反复冻融法破碎细胞。纯化后的Fab’抗体用SDS-PAGE电泳检测，结果显示抗体蛋白条带单一，非竞争性ELISA测定抗体与TNF-α的亲和常数为Ka=4×10-~(13)M~(-1)。另外体外中和试验显示，Fab’抗体可以在一定程度上中和TNF-α对L929细胞的杀伤作用，在浓度为3.2ng/ml时对TNF-α细胞毒的中和率达到85%，在800pg/ml时对TNF-α细胞毒的中和率达到48%。 本研究以原核表达的TNF-α为抗原，从噬菌体抗体库中筛选TNF-α抗体并对其进行Fab’形式改造，最终获得具有一定生物学活性的TNF-α人源性中和Fab’抗体，为相关疾病的治疗奠定良好的基础。
[Abstract]:Tumor necrosis factor - alpha (TNF- alpha) is a key cytokine in the pathological changes of rheumatoid arthritis (RA) and other diseases. The results of clinical trials have confirmed that TNF- alpha is a suitable target for the treatment of this disease. The inhibitors of TNF- alpha have achieved good results in the treatment of RA, and their representative drugs are Enbrel, Remicade, Humira and so on. The clinical trials of TNF antibody drugs have been developed with new indications and the market space is expanding. These drugs have the advantages of high specificity, good targeting and low side effects. They have gradually become the first-line drugs for the treatment of RA with methotrexate. But they also exist in different defects, including the following two points: first, Rem ICADE is human mouse chimeric antibody, which contains 25% mouse source fragments. Long-term injection may cause human anti mouse antibody (HAMA) reaction. Second, the Fc segments carried by these antibodies can activate complement and ADCC activity, which will lead to the expression of TNF cell apoptosis and produce a series of side effects. Therefore, it is particularly important to develop a whole human small molecule TNF- alpha antibody. Meaning.
The research and development of the TNF- alpha antibody of all human small molecules in this project includes three parts: expression and purification of TNF- alpha antigen, screening of antibody library and expression and purification of anti TNF- alpha Fab 'antibody.
The expression and purification of TNF- alpha antigen: according to the human TNF- alpha sequence in GenBank, the TNF- alpha gene was synthesized by the whole chemical synthesis, and the gene was extracted and transformed into the TNF- alpha expression vector pET-23b/TNF- A and transformed into the Escherichia coli BL21 (DE3) to obtain the positive clones. The positive clones were cloned at 37 C to express 16h to the soluble secretory expression of TNF- a in the supernatant of the fermentation liquid. After being concentrated and purified by chromatography, the purity of TNF- alpha was obtained. The purity was more than 95%, the specific activity was 4.4 * 10~6U/mg, and the yield was 4mg/L.
Antibody library screening: we use TNF- alpha as an antigen to screen the phage antibody library to obtain antibody clones that have specific affinity for TNF- alpha. After six rounds of solid phase scrub, the phage antibody clones with TNF- alpha specificity increased from 1.1 x 10~3pfu to 2 x 10~5pfu, and the output input ratio rises from 3.2 x 10~ (-8) to 1.2 x 10~ (-6). Although the elution conditions were more stringent with the increase of the number of screening wheels, 200 times of enrichment was finally obtained. 200 clones were selected randomly from sixth rounds of clones for ELISA screening and 5 positive clones were obtained. Further, the specificity of antigen binding was further compared to the 5 clones, and one of the most specific TNF- alpha specificity was determined. Good cloned G11.
The expression and purification of anti TNF- alpha Fab 'antibody: the screened G11 clones were sequenced, the antibody gene sequence was obtained, and the expression vector of Fab' antibody was constructed. After the vector was transformed into the Escherichia coli BL21 (DE3), the positive clones were obtained. The expression conditions were optimized and the effects of the medium and induction time on the expression were compared. The results showed that the expression of the antibody was compared. With the LB medium at 30 and IPTG induced concentration 0.1mM, the expression of 24h, Fab 'antibody was highest. We also compared the method of bacterial wall breaking. We selected the broken cells in ultrasonic, osmotic shock, lysozyme and repeated freezing and thawing. The purified Fab' antibody was detected by SDS-PAGE electrophoresis, and the result showed antibody protein. The affinity constant of the non competitive ELISA antibody and TNF- alpha was Ka=4 * 10-~ (13) M~ (-1). In addition, in vitro neutralization test showed that Fab 'antibody could neutralize the killing effect of TNF- a to L929 cells to a certain extent, and the neutralization rate of TNF- alpha cytotoxic at the concentration of 3.2ng/ml was 85%. And the rate is up to 48%.
In this study, the TNF- alpha expressed by prokaryotic expression was used as antigen to screen TNF- alpha antibody from phage antibody library and to transform it in Fab 'form. Finally, a certain biological activity of TNF- alpha human neutralization and Fab' antibody was obtained, which laid a good foundation for the treatment of related diseases.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2172642
[Abstract]:Tumor necrosis factor - alpha (TNF- alpha) is a key cytokine in the pathological changes of rheumatoid arthritis (RA) and other diseases. The results of clinical trials have confirmed that TNF- alpha is a suitable target for the treatment of this disease. The inhibitors of TNF- alpha have achieved good results in the treatment of RA, and their representative drugs are Enbrel, Remicade, Humira and so on. The clinical trials of TNF antibody drugs have been developed with new indications and the market space is expanding. These drugs have the advantages of high specificity, good targeting and low side effects. They have gradually become the first-line drugs for the treatment of RA with methotrexate. But they also exist in different defects, including the following two points: first, Rem ICADE is human mouse chimeric antibody, which contains 25% mouse source fragments. Long-term injection may cause human anti mouse antibody (HAMA) reaction. Second, the Fc segments carried by these antibodies can activate complement and ADCC activity, which will lead to the expression of TNF cell apoptosis and produce a series of side effects. Therefore, it is particularly important to develop a whole human small molecule TNF- alpha antibody. Meaning.
The research and development of the TNF- alpha antibody of all human small molecules in this project includes three parts: expression and purification of TNF- alpha antigen, screening of antibody library and expression and purification of anti TNF- alpha Fab 'antibody.
The expression and purification of TNF- alpha antigen: according to the human TNF- alpha sequence in GenBank, the TNF- alpha gene was synthesized by the whole chemical synthesis, and the gene was extracted and transformed into the TNF- alpha expression vector pET-23b/TNF- A and transformed into the Escherichia coli BL21 (DE3) to obtain the positive clones. The positive clones were cloned at 37 C to express 16h to the soluble secretory expression of TNF- a in the supernatant of the fermentation liquid. After being concentrated and purified by chromatography, the purity of TNF- alpha was obtained. The purity was more than 95%, the specific activity was 4.4 * 10~6U/mg, and the yield was 4mg/L.
Antibody library screening: we use TNF- alpha as an antigen to screen the phage antibody library to obtain antibody clones that have specific affinity for TNF- alpha. After six rounds of solid phase scrub, the phage antibody clones with TNF- alpha specificity increased from 1.1 x 10~3pfu to 2 x 10~5pfu, and the output input ratio rises from 3.2 x 10~ (-8) to 1.2 x 10~ (-6). Although the elution conditions were more stringent with the increase of the number of screening wheels, 200 times of enrichment was finally obtained. 200 clones were selected randomly from sixth rounds of clones for ELISA screening and 5 positive clones were obtained. Further, the specificity of antigen binding was further compared to the 5 clones, and one of the most specific TNF- alpha specificity was determined. Good cloned G11.
The expression and purification of anti TNF- alpha Fab 'antibody: the screened G11 clones were sequenced, the antibody gene sequence was obtained, and the expression vector of Fab' antibody was constructed. After the vector was transformed into the Escherichia coli BL21 (DE3), the positive clones were obtained. The expression conditions were optimized and the effects of the medium and induction time on the expression were compared. The results showed that the expression of the antibody was compared. With the LB medium at 30 and IPTG induced concentration 0.1mM, the expression of 24h, Fab 'antibody was highest. We also compared the method of bacterial wall breaking. We selected the broken cells in ultrasonic, osmotic shock, lysozyme and repeated freezing and thawing. The purified Fab' antibody was detected by SDS-PAGE electrophoresis, and the result showed antibody protein. The affinity constant of the non competitive ELISA antibody and TNF- alpha was Ka=4 * 10-~ (13) M~ (-1). In addition, in vitro neutralization test showed that Fab 'antibody could neutralize the killing effect of TNF- a to L929 cells to a certain extent, and the neutralization rate of TNF- alpha cytotoxic at the concentration of 3.2ng/ml was 85%. And the rate is up to 48%.
In this study, the TNF- alpha expressed by prokaryotic expression was used as antigen to screen TNF- alpha antibody from phage antibody library and to transform it in Fab 'form. Finally, a certain biological activity of TNF- alpha human neutralization and Fab' antibody was obtained, which laid a good foundation for the treatment of related diseases.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 林丽珠，陈春妹，喻红，吕群，陈晓龙，姚锡惠，陈海明，赵寿元;柱层析法纯化重组人肿瘤坏死因子α及其抑癌作用[J];复旦学报(自然科学版);1994年05期
2 张振龙,张琰平,宁杨,窦金福,陈梅娣,崔春青,赵秀芬,樊晓翔,倪道明,李昌本;重组人肿瘤坏死因子α的分离纯化及鉴定[J];中国生物制品学杂志;1998年02期
本文编号：2172642
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