本文關(guān)鍵詞：抗TNF-α多價(jià)抗體的構(gòu)建及體內(nèi)外生物學(xué)活性研究　出處：《中國(guó)協(xié)和醫(yī)科大學(xué)》2005年博士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：腫瘤壞死因子α(TNF-α)是上世紀(jì)70年代發(fā)現(xiàn)的一個(gè)細(xì)胞因子，最初發(fā)現(xiàn)它能引起某些特定的腫瘤組織的出血性壞死，現(xiàn)在已經(jīng)知道它除了具有抗腫瘤作用外，還在機(jī)體的免疫、炎癥和病理中發(fā)揮重要的作用。TNF-α的拮抗劑包括中和性的抗體已被證明是急性膿毒血癥、類(lèi)風(fēng)濕關(guān)節(jié)炎，局部性回腸炎和牛皮癬等急慢性炎癥性疾病的有效治療劑。美國(guó)FDA已批準(zhǔn)了Etanercept、Infliximab和Adalimumab這三種抗TNF-α的生物治療劑，其中后兩種為抗TNF-α的單克隆抗體。抗體工程技術(shù)經(jīng)歷了鼠源雜交瘤抗體、人-鼠嵌合抗體、人源化抗體和人源抗體的發(fā)展階段，而上世紀(jì)80年代基因工程技術(shù)的興起使構(gòu)建基因工程的小分子抗體如Fab、scFv及單域抗體等成為現(xiàn)實(shí)，這些小分子抗體由于分子量小、穿透性強(qiáng)、易于構(gòu)建并可在大腸桿菌等原核系統(tǒng)中功能性的表達(dá)而具有廣闊的應(yīng)用前景。本研究通過(guò)基因工程的方法構(gòu)建了具有中和活性的抗TNF-α的單鏈抗體，對(duì)其生物學(xué)活性進(jìn)行了研究，并在此基礎(chǔ)上對(duì)單鏈抗體進(jìn)行了多價(jià)化，對(duì)多價(jià)抗體的體內(nèi)外生物活性進(jìn)行了研究，為開(kāi)發(fā)新的抗TNF-α治療劑打下了基礎(chǔ)。 一、抗TNF-α單鏈抗體TNF-scFv的構(gòu)建及體外活性研究 單鏈抗體是一種將抗體的重鏈可變區(qū)(VH)和輕鏈可變區(qū)(VL)通過(guò)一段連接肽連接起來(lái)而形成的重組蛋白。本研究利用連接肽(GGGGS)_3將抗TNF-α的單克隆抗體Di62的VH和VL連接起來(lái)，設(shè)計(jì)成為完整的單鏈抗體的氨基酸序列，然后根據(jù)設(shè)計(jì)的氨基酸序列以大腸桿菌偏愛(ài)的密碼子將其反向翻譯成編碼的DNA序列。根據(jù)編碼的DNA序列，合成了22個(gè)寡核苷酸片段并用裝配PCR的方法將這22個(gè)寡核苷酸片段拼接成的完整的單鏈抗體編碼基因，在編碼基因的兩端加上了克隆所需要的酶切位點(diǎn)。將此編碼基因克隆進(jìn)載體pTCM-2，通過(guò)陽(yáng)性克隆的鑒定和序列測(cè)定，構(gòu)建了單鏈抗體TNF-scFv的表達(dá)載體，命名為pTS。將載體pTS轉(zhuǎn)化大腸桿菌BL21(DE3)star，，利用0.4mM的IPTG誘導(dǎo)目
[Abstract]:Tumor necrosis factor- 偽 (TNF- 偽) is a cytokine found in -30s, which was initially found to cause hemorrhagic necrosis in certain tumor tissues. It is now known that in addition to its antitumor effects, it also plays an important role in the body's immunity, inflammation and pathology. TNF- 偽 antagonists, including neutralizing antibodies, have been shown to be acute sepsis. An effective treatment for acute and chronic inflammatory diseases such as rheumatoid arthritis, local colitis and psoriasis. The United States FDA has approved Etanercept. Infliximab and Adalimumab are three biotherapeutic agents against TNF- 偽. The latter two are monoclonal antibodies against TNF- 偽. The antibody engineering technology has experienced the development of murine hybridoma antibody, human-mouse chimeric antibody, humanized antibody and human antibody. In -20s, the rise of genetic engineering technology made the construction of small molecular antibodies such as FabscFv and single-domain antibodies become a reality. These small molecular antibodies have high penetration due to their small molecular weight. It is easy to construct and can be expressed in E. coli and other prokaryotes. In this study, a neutralizing single-chain antibody (scFv) against TNF- 偽 was constructed by genetic engineering. The biological activity of the antibody was studied, and on the basis of this, the single-chain antibody was polyvalent, and the biological activity of the polyvalent antibody in vivo and in vitro was studied. It lays a foundation for the development of a new anti-TNF- 偽 therapeutic agent. 1. Construction and in vitro activity of anti-TNF- 偽 scFv TNF-scFv Single chain antibody (scFv) is a recombinant protein formed by linking the heavy chain variable region (VH) and the light chain variable region (VLH) of the antibody via a linked peptide. The VH and VL of Di62, a monoclonal antibody against TNF- 偽, were linked together. The amino acid sequence of scFv was designed to be complete, and then reverse translated into the encoded DNA sequence according to the designed amino acid sequence with the preferred codon of Escherichia coli. According to the encoded DNA sequence. Twenty-two oligonucleotide fragments were synthesized and the 22 oligonucleotide fragments were spliced into a complete single-chain antibody coding gene by assembling PCR. The encoding gene was cloned into the vector pTCM-2 and identified and sequenced by positive cloning. The expression vector of scFv TNF-scFv was constructed and named pTS.The vector pTS was transformed into Escherichia coli BL21DE3DE-star. the target was induced by 0.4mm IPTG.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 馬玲;抗CD22人源化基因工程多價(jià)微型抗體的初步表達(dá)[D];廣西大學(xué);2007年

本文編號(hào)：1399454