本文選題：百草枯 + 噬菌體展示��； 參考：《華中農(nóng)業(yè)大學(xué)》2012年碩士論文

【摘要】：百草枯(1,1’-二甲基-4,4'-二氯二吡啶,PQ)是一種在農(nóng)業(yè)上應(yīng)用廣泛的非選擇性除草劑,它的大量使用給世界農(nóng)業(yè)帶來貢獻(xiàn),同時它的高致死率也引起了全球的關(guān)注。全世界每年都有多起百草枯中毒事件的報道,隨著報道的增多,醫(yī)生和學(xué)者們也開始對它關(guān)注起來。百草枯中毒的致死率為90%以上,導(dǎo)致中毒的原因有很多,如皮膚接觸,意外吸入或吞食以及自主吞服,其中自殺性吞服百草枯是主要原因。由于百草枯毒性劇烈,并發(fā)癥較多且往往伴隨有不可逆的后遺癥,而且臨床上目前尚無特效解毒藥,因此具有特異性以及高親和力的百草枯抗體的研究很有必要。人源非免疫抗體由于不會產(chǎn)生或只會有很小的免疫反應(yīng)而受到關(guān)注。 噬菌體展示抗體庫技術(shù)由于無需免疫動物,可直接從噬菌體抗體庫中淘選出特異噬菌體抗體,可以得到按常規(guī)免疫方法難以獲得的人源抗體,且所展示的抗體與其所對應(yīng)的基因存在于同一個重組噬菌體內(nèi),獲得抗體的同時也就獲得了它的基因,便于用抗體工程大規(guī)模生產(chǎn)抗體。因此可作人源抗體庫的理想載體。 本研究的目的是制備特異性中和百草枯的單克隆抗體。實驗部分主要包括全套人源抗體基因的克隆,抗體庫的構(gòu)建、動物免疫、抗體庫的篩選、單克隆抗體的表達(dá)、純化。 研究內(nèi)容主要有以下幾個方面： 將噬菌體的外殼蛋白10A在BLT5615中表達(dá)出來,純化后免疫家兔獲得用于T7噬菌體展示篩選的抗噬菌體外殼蛋白抗體,為T7噬菌體展示人源抗體庫篩查抗百草枯抗體奠定基礎(chǔ)。 從健康志愿者外周血中分離淋巴細(xì)胞,用Trizol法提取總RNA,用M-MLV逆轉(zhuǎn)錄酶反轉(zhuǎn)mRNA成cDNA后分別克隆人全套抗體重鏈可變區(qū)(VH)、輕鏈可變區(qū)(VL)基因,并利用重疊延伸PCR技術(shù)重組連接成抗體庫構(gòu)建所需要的VH-linker-VL單鏈抗體基因。經(jīng)EcoRⅠ和HindⅢ酶切后與T7噬菌體載體相連,完成體外包裝后,侵染宿主BLT5403擴(kuò)增得到初級庫。 采用固相篩選與液相篩選交替進(jìn)行的方式從人源抗體庫中篩選抗百草枯抗體。固相篩選：用OVA/BSA偶聯(lián)的百草枯作為抗原包被在固相支持物上,加入抗體庫孵育一定時間,洗去未結(jié)合的噬菌體從而富集特異噬菌體。液相篩選：羧基化的百草枯小分子與氨基化的磁珠偶聯(lián),加入抗體庫孵育一定時間,洗去未結(jié)合的噬菌體從而篩選特異性結(jié)合的噬菌體。 第一輪和第三輪用磁珠篩選,第二輪和第四輪分別用BSA偶聯(lián)的百草及枯OVA偶聯(lián)的百草枯包被固相載體篩選。經(jīng)過4輪“吸附—洗脫—擴(kuò)增”篩選過程,獲得抗原特異性較強(qiáng)的百草枯scFv噬菌體抗體。擴(kuò)增特異性較強(qiáng)的噬菌體抗體株中的目的片段,構(gòu)建表達(dá)載體進(jìn)行表達(dá)并純化抗體,對可溶性抗體的結(jié)合和競爭活性進(jìn)行驗證。 本實驗制備了T7噬菌體衣殼蛋白多克隆抗體,為噬菌體展示篩選特異性抗體實驗提供了基礎(chǔ)。成功構(gòu)建了庫容較大,多樣性好的T7Select單鏈抗體庫,為篩選抗多種單鏈抗體奠定了基礎(chǔ)。利用百草枯為抗原,直接從非免疫人源噬菌體抗體庫中篩選噬菌體抗體克隆,表達(dá)之后得到1株具有較好抑制率的特異性抗體,由于非免疫人源噬菌體抗體庫的庫容非常大,而噬菌體本身對BSA以及OVA有一定的結(jié)合能力,不能排除與封閉液等的非特異性結(jié)合的可能性,進(jìn)而增加了淘洗的難度和非特異性抗體的產(chǎn)生。因此,仍需完善和探索更好的實驗方法。
[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 .

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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