T7噬菌体展示人源抗体库筛选百草枯抗体

发布时间：2018-05-15 19:33

本文选题：百草枯 + 噬菌体展示　；参考：《华中农业大学》2012年硕士论文

【摘要】：百草枯(1,1’-二甲基-4,4'-二氯二吡啶,PQ)是一种在农业上应用广泛的非选择性除草剂,它的大量使用给世界农业带来贡献,同时它的高致死率也引起了全球的关注。全世界每年都有多起百草枯中毒事件的报道,随着报道的增多,医生和学者们也开始对它关注起来。百草枯中毒的致死率为90%以上,导致中毒的原因有很多,如皮肤接触,意外吸入或吞食以及自主吞服,其中自杀性吞服百草枯是主要原因。由于百草枯毒性剧烈,并发症较多且往往伴随有不可逆的后遗症,而且临床上目前尚无特效解毒药,因此具有特异性以及高亲和力的百草枯抗体的研究很有必要。人源非免疫抗体由于不会产生或只会有很小的免疫反应而受到关注。噬菌体展示抗体库技术由于无需免疫动物,可直接从噬菌体抗体库中淘选出特异噬菌体抗体,可以得到按常规免疫方法难以获得的人源抗体,且所展示的抗体与其所对应的基因存在于同一个重组噬菌体内,获得抗体的同时也就获得了它的基因,便于用抗体工程大规模生产抗体。因此可作人源抗体库的理想载体。本研究的目的是制备特异性中和百草枯的单克隆抗体。实验部分主要包括全套人源抗体基因的克隆,抗体库的构建、动物免疫、抗体库的筛选、单克隆抗体的表达、纯化。研究内容主要有以下几个方面：将噬菌体的外壳蛋白10A在BLT5615中表达出来,纯化后免疫家兔获得用于T7噬菌体展示筛选的抗噬菌体外壳蛋白抗体,为T7噬菌体展示人源抗体库筛查抗百草枯抗体奠定基础。从健康志愿者外周血中分离淋巴细胞,用Trizol法提取总RNA,用M-MLV逆转录酶反转mRNA成cDNA后分别克隆人全套抗体重链可变区(VH)、轻链可变区(VL)基因,并利用重叠延伸PCR技术重组连接成抗体库构建所需要的VH-linker-VL单链抗体基因。经EcoRⅠ和HindⅢ酶切后与T7噬菌体载体相连,完成体外包装后,侵染宿主BLT5403扩增得到初级库。采用固相筛选与液相筛选交替进行的方式从人源抗体库中筛选抗百草枯抗体。固相筛选：用OVA/BSA偶联的百草枯作为抗原包被在固相支持物上,加入抗体库孵育一定时间,洗去未结合的噬菌体从而富集特异噬菌体。液相筛选：羧基化的百草枯小分子与氨基化的磁珠偶联,加入抗体库孵育一定时间,洗去未结合的噬菌体从而筛选特异性结合的噬菌体。第一轮和第三轮用磁珠筛选,第二轮和第四轮分别用BSA偶联的百草及枯OVA偶联的百草枯包被固相载体筛选。经过4轮“吸附—洗脱—扩增”筛选过程,获得抗原特异性较强的百草枯scFv噬菌体抗体。扩增特异性较强的噬菌体抗体株中的目的片段,构建表达载体进行表达并纯化抗体,对可溶性抗体的结合和竞争活性进行验证。本实验制备了T7噬菌体衣壳蛋白多克隆抗体,为噬菌体展示筛选特异性抗体实验提供了基础。成功构建了库容较大,多样性好的T7Select单链抗体库,为筛选抗多种单链抗体奠定了基础。利用百草枯为抗原,直接从非免疫人源噬菌体抗体库中筛选噬菌体抗体克隆,表达之后得到1株具有较好抑制率的特异性抗体,由于非免疫人源噬菌体抗体库的库容非常大,而噬菌体本身对BSA以及OVA有一定的结合能力,不能排除与封闭液等的非特异性结合的可能性,进而增加了淘洗的难度和非特异性抗体的产生。因此,仍需完善和探索更好的实验方法。
[Abstract]:Parcupine ( 1,1 ' - dimethyl - 4,4 ' - dichlorobipyridine , PQ ) is a widely used non - selective herbicide in agriculture , and its high fatality rate has attracted worldwide attention . With the increase of reports , doctors and scholars have begun to pay attention to it . The cause of poisoning is more than 90 % .

The phage display antibody library technology can be used for directly extracting specific phage antibodies from the phage antibody library without immunizing animal , and the humanized antibody which is difficult to obtain according to the conventional immunization method can be obtained , and the displayed antibody and the corresponding gene thereof are in the same recombinant bacteriophage , so that the antibody is obtained , and the gene thereof is obtained , so that the antibody can be produced on a large scale by using the antibody , and thus an ideal carrier of the human antibody library can be used .

The purpose of this study was to prepare a monoclonal antibody which was specific for the neutralization of parafterex . The experimental part mainly consisted of the cloning of human antibody gene , the construction of antibody library , animal immunity , the screening of antibody library , the expression and purification of monoclonal antibody .

The study mainly includes the following aspects :

The phage coat protein 10A is expressed in BLT5615 , and the purified immune rabbit obtains the anti - phage coat protein antibody for the T7 phage display screen , and lays a foundation for screening the anti - 100 - grass antibody library for the T7 phage display humanized antibody library .

Lymphocytes were isolated from peripheral blood of healthy volunteers . Total RNA was extracted by Trizol method . The VH and VL genes were cloned by reverse transcriptase of M - MLV reverse transcriptase mRNA . The VH - linker - VL single - chain antibody gene was constructed by overlapping extended PCR . After digestion with EcoRI and HindIII , the VH - linker - VL single - chain antibody gene was constructed .

The method of solid phase screening and liquid phase screening was used to screen the anti - cumyl antibody from human antibody library . Solid - phase screening was carried out with OVA / BSA as antigen coating on the solid support . The antibody library was added for incubation for a certain time . The unbound phage was added to enrich the specific phage . The liquid phase was screened : the carboxymethylated parafei small molecule was coupled with the amino magnetic beads , then the unbound phage was added to wash the unbound phage to screen the specific binding phage .

The first round and the third round are screened by magnetic beads , and the second and fourth wheels are respectively screened by BSA - coupled 100 - grass and dried OVA - coupled parafei . After four rounds of " adsorption - elution - amplification " screening process , the antibody is obtained with strong specificity in antigen specificity . The target fragment in the phage antibody strain with strong specificity is amplified , the expression vector is constructed to express and purify the antibody , and the binding and the competitive activity of the soluble antibody are verified .

In this experiment , the polyclonal antibody of T7 phage coat protein was prepared , which provided the basis for phage display and screening specific antibody experiments . The phage antibody library was successfully constructed by screening phage antibody library from non - immunized human phage antibody library .

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

【参考文献】