基于单域重链抗体的天然噬菌体文库的构建及抗DON适配体的淘选
发布时间：2019-06-17 19:18
【摘要】：重链抗体是一种存在于骆驼、鲨鱼等动物中天然缺失轻链的抗体,仅由其可变区组成的抗体称为单域重链抗体。单域重链抗体分子量小(约15 kDa),具有易表达、高水溶性、耐高温、耐极度pH值等独特性质,目前已广泛用于基础研究、医学诊断和检测、抗体药物开发等领域。在食品安全检测领域单域重链抗体也展现出广阔的发展前景。 脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON),又名呕吐毒素(vomitoxin,VT),属于单端孢霉烯族真菌毒素,主要是由某些镰刀菌产生。该毒素常出现于谷物及其加工产品中,许多国家和地区都检测到了DON的污染。DON会引起动物增重下降、食欲减退、营养吸收率降低,并影响免疫功能。目前大部分国家对食品、谷物、饲料中的DON含量都做了严格规定。 免疫学检测方法具有灵敏、快速等特点,适合大批量样本的检测。现有的DON免疫学检测方法大多采用传统的抗体制备技术(多克隆抗体和单克隆抗体),需要多次反复免疫动物,制备过程繁琐,来源有限,生产成本较高。以抗体库技术为代表的第三代基因工程抗体技术为快速、低成本的获取和生产抗体提供了强有力的支撑。 本研究采用未经免疫的健康羊驼(Lama pacos)的免疫细胞作为实验材料,根据重链抗体保守序列设计引物,建立基于重链抗体可变区基因的天然噬菌体展示文库,并以DON-MBSA人工抗原为配基,对构建的文库进行了淘选,主要研究结果如下： 1以羊驼(Lama pacos)外周血为起始材料,分别采用半巢式PCR法和巢式PCR法构建了两个单域重链抗体初级文库SNAL和NAL,两者的实际库容量均达到107,经辅助噬菌体救援后获得噬菌体展示文库SNA-PDL和NA-PDL,滴度达1013cfu/mL。文库多样性分析结果显示,文库具有良好的多样性,可以用于后续淘选。 2通过三轮淘选,从噬菌体文库SNA-PDL中淘选出了三类能与DON特异性结合的克隆,分别为DON-binder 1:B9、B10、B11、B13、C3、C8、C10、C15; DON-binder 2:C11; DON-binder 3:A4、A9。间接phage-ELISA检测结果显示,前两类克隆的OD450测定值可达后一类克隆OD450值的7～10倍,提示亲和力较高。从天然噬菌体文库NA-PDL中淘选获得了两种可以与DON-MBSA结合的单域重链抗体,其中一种可以与DON特异性结合。 3在大肠杆菌表达系统中对重组噬菌粒pHEN-B9进行诱导表达,SDS-PAGE电泳结果显示,在诱导培养上清中存在预期大小的蛋白(约50 kDa)。竞争ELISA法分析诱导表达上清,结果表明诱导表达上清可以抑制抗DON单克隆抗体与人工抗原的结合,提示培养上清中的表达产物能与DON发生特异性结合,有望替代抗DON单克隆抗体。 本研究的主要创新之处： 1采用针对羊驼(Lama pacos)重链抗体设计的引物,分别以巢式PCR法和半巢式PCR法扩增羊驼重链抗体可变区全套基因,构建了两个天然噬菌体文库SNA-PDL和NA-PDL,该文库可以作为通用的技术平台,重复用于多种抗原的筛选。 2从天然噬菌体文库SNA-PDL中淘选获得了三种可以与DON特异结合的克隆,选取其中一种结合力较强的克隆在大肠杆菌中表达,结果表明,能够表达出具有DON结合活性的蛋白,有望将其应用于DON免疫学检测方法的建立。从天然噬菌体文库NA-PDL中淘选获得了一种可以与DON特异性结合的单域重链抗体。
[Abstract]:The heavy chain antibody is an antibody that is naturally missing a light chain in an animal such as a camel, a shark, and the like, and an antibody composed of only a variable region thereof is referred to as a single-domain heavy chain antibody. The single-domain heavy chain antibody has a small molecular weight (about 15 kDa), and has unique properties such as easy expression, high water solubility, high temperature resistance, extreme pH value, and the like, and is widely used in the fields of basic research, medical diagnosis and detection, antibody drug development and the like. The single-domain heavy chain antibody in the field of food safety detection also has a wide development prospect. Deoxynivalenol (DON), also known as a Vomitoxin (VT), belongs to a single-end trichoderma mycotoxin, which is mainly caused by certain fusarium The toxin is often present in the grain and its processed products, and DON is detected in many countries and regions Contamination. DON causes decreased weight gain, decreased appetite, reduced nutritional absorption, and immune response In most countries, the DON content in food, grain and feed is strictly controlled. The immunological detection method is sensitive, rapid and the like, and is suitable for mass production. The conventional DON immunological detection method mainly adopts the traditional antibody preparation technology (polyclonal antibody and monoclonal antibody), The third-generation genetic engineering antibody technology, which is represented by the antibody library technology, is a rapid, low-cost acquisition and production antibody. In this study, the immune cells of the unimmunized healthy alpaca (Lama pacos) were used as the experimental material. The primers were designed according to the conservative sequence of the heavy chain antibody, and a natural phage display library based on the variable region gene of the heavy chain was established and the DON-MBS was used. A artificial antigen is a ligand, and the constructed library is subjected to panning, The main results are as follows:1. The peripheral blood of the Lama pacos is used as the starting material, and the two single-domain heavy chain antibody primary libraries, NAL and NAL, are constructed by using a semi-nested PCR method and a nested PCR method, respectively. The actual library capacity reached 107, and the SNA-PDL and NA-PDL, titer of the phage display library were obtained after the auxiliary phage rescue. The results of the analysis of the library diversity showed that the library had a good multi-function. Samples can be used for subsequent panning. Three types of clones capable of specifically binding to DON are selected from the phage library, SNA-PDL, by three-wheel panning, respectively: DON-finder 1: B9, B10, B11, B13, C3, C8, C10, C15; DON-Binder 2: C11; DON -Binder 3: A4, A9. The indirect phage-ELISA test results showed that the OD450 values of the first two types of clones can reach the last class of clone OD450 values Two single-domain heavy chain antibodies which can be combined with DON-MBSA are obtained from the natural phage library NA-PDL, and the single-domain heavy chain antibody can be combined with DON-MBSA, in that expression system of E. coli, the recombinant phagemid pHen-B9 is induce and expressed, and the SDS-PAGE electrophoresis result shows that in the culture supernatant, The protein (about 50 kDa) of the expected size was present. The expression of the supernatant was analyzed by competitive ELISA. The results showed that the expression of the supernatant could inhibit the binding of the anti-DON monoclonal antibody to the artificial antigen, suggesting that the expression product in the culture supernatant can be specific to DON. Sex-binding, promising alternative to anti-DO The main innovations of this study were:1. Using the primers designed for the heavy chain antibody of the alpaca (Lama pacos), the whole gene of the variable region of the heavy chain of the alpaca was amplified by a nested PCR method and a semi-nested PCR method. Two natural phage libraries, SNA-PDL and NA-PDL, the library Can be used as a general technical platform and is repeatedly used for screening a plurality of antigens. Three clones which can be specifically combined with DON are obtained from the SNA-PDL of the natural phage library, and one of the clones with strong binding force is selected to be in Escherichia coli. The results show that the combination of DON and DON can be expressed. The active protein is expected to be applied to the establishment of the DON immunological detection method. From the natural phage library, NA-PDL
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
[Abstract]:The heavy chain antibody is an antibody that is naturally missing a light chain in an animal such as a camel, a shark, and the like, and an antibody composed of only a variable region thereof is referred to as a single-domain heavy chain antibody. The single-domain heavy chain antibody has a small molecular weight (about 15 kDa), and has unique properties such as easy expression, high water solubility, high temperature resistance, extreme pH value, and the like, and is widely used in the fields of basic research, medical diagnosis and detection, antibody drug development and the like. The single-domain heavy chain antibody in the field of food safety detection also has a wide development prospect. Deoxynivalenol (DON), also known as a Vomitoxin (VT), belongs to a single-end trichoderma mycotoxin, which is mainly caused by certain fusarium The toxin is often present in the grain and its processed products, and DON is detected in many countries and regions Contamination. DON causes decreased weight gain, decreased appetite, reduced nutritional absorption, and immune response In most countries, the DON content in food, grain and feed is strictly controlled. The immunological detection method is sensitive, rapid and the like, and is suitable for mass production. The conventional DON immunological detection method mainly adopts the traditional antibody preparation technology (polyclonal antibody and monoclonal antibody), The third-generation genetic engineering antibody technology, which is represented by the antibody library technology, is a rapid, low-cost acquisition and production antibody. In this study, the immune cells of the unimmunized healthy alpaca (Lama pacos) were used as the experimental material. The primers were designed according to the conservative sequence of the heavy chain antibody, and a natural phage display library based on the variable region gene of the heavy chain was established and the DON-MBS was used. A artificial antigen is a ligand, and the constructed library is subjected to panning, The main results are as follows:1. The peripheral blood of the Lama pacos is used as the starting material, and the two single-domain heavy chain antibody primary libraries, NAL and NAL, are constructed by using a semi-nested PCR method and a nested PCR method, respectively. The actual library capacity reached 107, and the SNA-PDL and NA-PDL, titer of the phage display library were obtained after the auxiliary phage rescue. The results of the analysis of the library diversity showed that the library had a good multi-function. Samples can be used for subsequent panning. Three types of clones capable of specifically binding to DON are selected from the phage library, SNA-PDL, by three-wheel panning, respectively: DON-finder 1: B9, B10, B11, B13, C3, C8, C10, C15; DON-Binder 2: C11; DON -Binder 3: A4, A9. The indirect phage-ELISA test results showed that the OD450 values of the first two types of clones can reach the last class of clone OD450 values Two single-domain heavy chain antibodies which can be combined with DON-MBSA are obtained from the natural phage library NA-PDL, and the single-domain heavy chain antibody can be combined with DON-MBSA, in that expression system of E. coli, the recombinant phagemid pHen-B9 is induce and expressed, and the SDS-PAGE electrophoresis result shows that in the culture supernatant, The protein (about 50 kDa) of the expected size was present. The expression of the supernatant was analyzed by competitive ELISA. The results showed that the expression of the supernatant could inhibit the binding of the anti-DON monoclonal antibody to the artificial antigen, suggesting that the expression product in the culture supernatant can be specific to DON. Sex-binding, promising alternative to anti-DO The main innovations of this study were:1. Using the primers designed for the heavy chain antibody of the alpaca (Lama pacos), the whole gene of the variable region of the heavy chain of the alpaca was amplified by a nested PCR method and a semi-nested PCR method. Two natural phage libraries, SNA-PDL and NA-PDL, the library Can be used as a general technical platform and is repeatedly used for screening a plurality of antigens. Three clones which can be specifically combined with DON are obtained from the SNA-PDL of the natural phage library, and one of the clones with strong binding force is selected to be in Escherichia coli. The results show that the combination of DON and DON can be expressed. The active protein is expected to be applied to the establishment of the DON immunological detection method. From the natural phage library, NA-PDL
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 谢磊,孙建波,张世清,黄俊生;大肠杆菌表达系统及其研究进展[J];华南热带农业大学学报;2004年02期
2 冯强,杨s，
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