構(gòu)建大容量噬菌體抗體庫
發(fā)布時(shí)間：2019-06-11 18:33
【摘要】： 噬菌體抗體庫是把體外隨機(jī)組合的全套抗體基因呈現(xiàn)在絲狀噬菌體表面,通過抗原的親和力選擇和噬菌體擴(kuò)增獲得特異性抗體基因的技術(shù)。該技術(shù)已廣泛用于制備人源單克隆抗體及免疫學(xué)研究中。 本研究通過構(gòu)建噬粒載體并利用噬菌體抗體庫技術(shù)和Cre-Loxp定位重組系統(tǒng),構(gòu)建了一個(gè)大容量天然噬菌體抗體庫。我們采集了120人份正常人的外周血,分離淋巴細(xì)胞,提取細(xì)胞總RNA反轉(zhuǎn)錄cDNA,分別以不同的人源抗體基因引物擴(kuò)增抗體輕、重鏈基因。首先將輕鏈基因按照人類抗體基因使用頻率的比例混和,插入到PDF-D-SacB質(zhì)粒中,通過多次電轉(zhuǎn)化,收獲含有輕鏈庫的質(zhì)粒；然后將重鏈基因按照人類抗體基因使用頻率的比例混合后,插入到輕鏈質(zhì)粒獲得初級(jí)非免疫抗體庫。初級(jí)噬菌體抗體庫以高感染復(fù)數(shù)(MOI100：1)感染能分泌重組酶的大腸桿菌BS1365,使多個(gè)噬菌體同時(shí)感染一個(gè)細(xì)菌,30℃低溫誘導(dǎo)定點(diǎn)重組,即VL與VH發(fā)生重組,收獲的重組噬菌體抗體庫以低感染復(fù)述(MOI100：1)感染大腸桿菌Trans1-Blue,收獲噬菌體獲得含有1.0×1011克隆的噬菌體抗體庫,隨機(jī)挑取10個(gè)克隆鑒定,輕鏈和重鏈均有插入的10個(gè)克隆,插入率為100%。 通過以上實(shí)驗(yàn)得到了以下結(jié)論：本研究獲得所有VL和VH亞類基因,擴(kuò)增產(chǎn)物片段大小均與理論值相符；利用噬菌體抗體庫技術(shù)和Cre-Loxp定位重組系統(tǒng),構(gòu)建了一個(gè)大容量的人源天然噬菌體抗體庫；輕重鏈基因的克隆效率均為100%。初級(jí)庫容量為7.2×1012,滴度為6×1013；重組后的工作庫有效容量為1.0×1011,滴度至少為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，
本文編號(hào)：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，
本文編號(hào)：2497384
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