天然人源噬菌体抗体库的构建与初步筛选

发布时间：2018-01-08 08:31

本文关键词：天然人源噬菌体抗体库的构建与初步筛选　出处：《北京生物制品研究所》2009年硕士论文　论文类型：学位论文

【摘要】： 将抗体的多样性可变区基因组装到表达载体内,表达到噬菌体表面,得到多样性噬菌体抗体的集合即为噬菌体抗体库。这一技术基于噬菌体表面展示技术的发展,相比较于多克隆抗体血清和B细胞杂交瘤技术,带来了抗体工程技术的一次巨大革命。它不仅解决了人源抗体制备的问题,更使得对抗体性能的改良进入了新阶段,可以筛选出具有特定性能的未知结构。其中,大容量通用性噬菌体抗体库的构建和筛选技术的不断改进,为高通量快速筛选特异性抗体提供了简便和高效的可操作系统,使得不经免疫既可获得各种特异性抗体。噬菌体抗体库分为合成抗体库、半合成抗体库以及天然库(免疫或非免疫库)。免疫抗体库的抗体基因来自于经抗原免疫的个体,易于获得针对该特定抗原的抗体克隆,这类文库具有较强的抗原特异性和亲和力。相比较于免疫库,更为理想的是构建无免疫偏向性的抗体库,可以同时针对多种人类抗原进行筛选。通过收获分离正常人外周血淋巴细胞、脾细胞、骨髓细胞中的mRNA,扩增抗体基因并克隆到噬菌体载体后形成的天然非免疫性通用性抗体库含有大量识别不同抗原的抗体克隆。与B细胞杂交瘤技术和免疫文库相比,其具有许多优点:包含巨大数量的多样性的抗体克隆;可以分离出针对那些不引起机体免疫反应的弱抗原、自身抗原和具有毒性抗原的抗体;一个抗体库可以用于多种抗原筛选而获得多种抗体产生;在库容量足够大的时候可以获得高亲和力抗体。但是非免疫库也存在一些局限性:在库容量较小时分离到低亲和力抗体;构建抗体库时间此较长;抗体库的质量和大小受可变区基因表达情况的影响;B细胞捐赠者的免疫历史不清楚和IgM库潜在多样性的限制。本研究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,用眼睛蛇毒和HCV抗原分别对噬菌体抗体库进行筛选以获得人源抗体。为构建具有良好多样性的抗体库,我们采集了150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(K:60%λ:40%HVK1:19.2%HVK2:10.2%HVK3:30.6%HVL1:14.8%HVL2:13.2%HVL3:9.2%),插入到pDF质粒中,通过多次电转化,铺板刮细菌收获含有轻链库质粒,然后将重链按照人类抗体基因使用频率的比例(HVH1:38%HVH2:34%HVH3:2%HVH4:26%),混合后插入轻链质粒获得含有8.8×10~8克隆的初级非免疫抗体库。初级噬菌体抗体库以高感染复数(MOI＞100:1)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,低温诱导定点重组,收获的重组噬菌体抗体库以低感染复述(MOI＜1:1)感染大肠杆菌XL1-Blue,噬菌体的基因型和表型得到统一,收获噬菌体获得含有9×10~(10)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8个克隆,插入率为80%。随机挑选96个克隆测序,获得的序列结果表明没有重复的序列,序列分析表明轻重链各亚型分布基本符合人类抗体基因使用频率的比例。用HCV NS4和Cobra venom对天然库进行了4轮富集筛选,4轮富集筛选的富集效果明显。随机挑选4轮富集筛选后的单克隆诱导表达后的上清进行初步检测。随机挑选4轮富集筛选后的单克隆诱导表达后的上清进行初步检测。用96孔板进行高通量的克隆筛选,获得了20株具有Fab活性的抗体克隆,1株特异性的针对HCV NS4,一株针对Cobra venom,筛选结果并不理想,还需要进一步调整实验条件来获得特异性的抗体克隆。本研究利用噬菌体抗体库技术,采集多份正常健康人外周血利用Cre-Loxp定位重组系统,构建了大容量的天然噬菌体抗体库并对抗体库的序列分布进行了分析;并用眼镜蛇毒和HCV抗原,对抗体库进行初步筛选,获得了20株具有Fab活性的抗体克隆,1株特异性的针对眼镜蛇毒抗原,1株特异性的针对HCV抗原,但获得的抗体的反应值较低,有必要对抗体库进行进一步的改进和调整筛选的方法和条件。
[Abstract]:This technique is based on the development of phage surface display technology . This technique is based on the development of phage surface display technology . This technique is based on the development of phage surface display technology . It has brought about a great revolution of anti - body engineering technology . It not only solves the problem of human antibody preparation , but also makes it possible to filter out the unknown structure with specific properties . A phage antibody library is divided into a synthetic antibody library , a semi - synthetic antibody library and a natural library ( immune or non - immune library ) . The antibody gene of the immune antibody library is derived from an individual immunized with an antigen and is easy to obtain an antibody clone aiming at the specific antigen . In order to construct an antibody library with good diversity , we collected 150 normal human peripheral blood , isolated lymphocytes , extracted the total RNA reverse transcribed cDNA of human antibody , and then inserted into the pDF plasmid . Then , the heavy chain was inserted into pDF plasmid . Then the heavy chain was inserted into the pDF plasmid . Then the heavy chain was inserted into pDF plasmid . Then the heavy chain was inserted into pDF plasmid . Then the heavy chain was inserted into the plasmid of human antibody gene ( HVH1 : 38 % HVH2 : 34 % HVH3 : 2 % HVH4 : 26 % ) . Then the light chain plasmid was inserted into the light chain plasmid to obtain the primary non - immune antibody library containing 8.8 脳 10 ~ 8 clones . coli XL1 - Blue was infected with a phage antibody library containing 9 脳 10 ~ ( 10 ) clones . The recombinant phage antibody library with 9 脳 10 ~ ( 10 ) clones was obtained by random selection of 96 clones . The results showed that there were no repeated sequences , and the sequence analysis showed that the distribution of each sub - subtype of heavy chain was basically in accordance with the frequency of human antibody gene use . Four rounds of enrichment and screening were carried out with HCV NS4 and Cobra venom , and the enrichment effect of 4 rounds of enrichment and screening was obvious . The supernatant after 4 rounds of enrichment and screening was selected randomly . In this study , using phage antibody library technique , a large - capacity natural phage antibody library was collected and the sequence distribution of the antibody library was analyzed . The antibody library was preliminarily screened by using Cobra venom and HCV antigen . One specific antibody clone with Fab activity was obtained . One specific antibody was specific for HCV antigen , but the antibody response was low , and it was necessary to further improve and adjust the antibody library .

【学位授予单位】：北京生物制品研究所
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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