构建大容量噬菌体抗体库
发布时间：2019-06-11 18:33
【摘要】： 噬菌体抗体库是把体外随机组合的全套抗体基因呈现在丝状噬菌体表面,通过抗原的亲和力选择和噬菌体扩增获得特异性抗体基因的技术。该技术已广泛用于制备人源单克隆抗体及免疫学研究中。 本研究通过构建噬粒载体并利用噬菌体抗体库技术和Cre-Loxp定位重组系统,构建了一个大容量天然噬菌体抗体库。我们采集了120人份正常人的外周血,分离淋巴细胞,提取细胞总RNA反转录cDNA,分别以不同的人源抗体基因引物扩增抗体轻、重链基因。首先将轻链基因按照人类抗体基因使用频率的比例混和,插入到PDF-D-SacB质粒中,通过多次电转化,收获含有轻链库的质粒；然后将重链基因按照人类抗体基因使用频率的比例混合后,插入到轻链质粒获得初级非免疫抗体库。初级噬菌体抗体库以高感染复数(MOI100：1)感染能分泌重组酶的大肠杆菌BS1365,使多个噬菌体同时感染一个细菌,30℃低温诱导定点重组,即VL与VH发生重组,收获的重组噬菌体抗体库以低感染复述(MOI100：1)感染大肠杆菌Trans1-Blue,收获噬菌体获得含有1.0×1011克隆的噬菌体抗体库,随机挑取10个克隆鉴定,轻链和重链均有插入的10个克隆,插入率为100%。 通过以上实验得到了以下结论：本研究获得所有VL和VH亚类基因,扩增产物片段大小均与理论值相符；利用噬菌体抗体库技术和Cre-Loxp定位重组系统,构建了一个大容量的人源天然噬菌体抗体库；轻重链基因的克隆效率均为100%。初级库容量为7.2×1012,滴度为6×1013；重组后的工作库有效容量为1.0×1011,滴度至少为1.0×1013。
[Abstract]:Bacteriophage antibody library is a technique by which a complete set of antibody genes randomly combined in vitro is presented on the surface of filamentous bacteriophage, and the specific antibody gene is obtained by antigen affinity selection and bacteriophage amplification. This technique has been widely used in the preparation of human monoclonal antibodies and immunology. In this study, a large capacity natural bacteriophage antibody library was constructed by constructing macrophage vector and using bacteriophage antibody library technology and Cre-Loxp localization and recombination system. We collected 120 normal human peripheral blood, isolated lymphocytes, and extracted total RNA reverse transcriptional cDNA, to amplify antibody light and heavy chain genes with different human antibody gene primers. Firstly, the light chain gene was mixed into PDF-D-SacB plasmid according to the proportion of human antibody gene usage frequency, and the plasmid containing light chain library was obtained by multiple electrotransformation, and then the heavy chain gene was mixed according to the proportion of human antibody gene usage frequency and inserted into the light chain plasmid to obtain the primary non-immune antibody library. The primary bacteriophage antibody library infected E. coli BS1365, which could secrete recombinant enzyme with high infection complex (MOI100:1), infected multiple bacteriophages at the same time, and induced fixed-point recombination at 30 鈩，
本文编号：2497384
[Abstract]:Bacteriophage antibody library is a technique by which a complete set of antibody genes randomly combined in vitro is presented on the surface of filamentous bacteriophage, and the specific antibody gene is obtained by antigen affinity selection and bacteriophage amplification. This technique has been widely used in the preparation of human monoclonal antibodies and immunology. In this study, a large capacity natural bacteriophage antibody library was constructed by constructing macrophage vector and using bacteriophage antibody library technology and Cre-Loxp localization and recombination system. We collected 120 normal human peripheral blood, isolated lymphocytes, and extracted total RNA reverse transcriptional cDNA, to amplify antibody light and heavy chain genes with different human antibody gene primers. Firstly, the light chain gene was mixed into PDF-D-SacB plasmid according to the proportion of human antibody gene usage frequency, and the plasmid containing light chain library was obtained by multiple electrotransformation, and then the heavy chain gene was mixed according to the proportion of human antibody gene usage frequency and inserted into the light chain plasmid to obtain the primary non-immune antibody library. The primary bacteriophage antibody library infected E. coli BS1365, which could secrete recombinant enzyme with high infection complex (MOI100:1), infected multiple bacteriophages at the same time, and induced fixed-point recombination at 30 鈩，
本文编号：2497384
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