【摘要】：單鏈抗體(Single chain FV, scFV)是一種應(yīng)用最廣泛的小分子重組抗體,它是由一段彈性連接鏈(linker)把抗體重鏈可變區(qū)VH與輕鏈可變區(qū)VL相連而成,具有親代抗體的全部抗原結(jié)合特異性的最小功能結(jié)構(gòu)單位,大小為完整抗體的六分之一。它有分子小、穿透力強(qiáng)、體內(nèi)半衰期短、免疫原性低,可通過(guò)包涵體大量表達(dá)、易于基因操作,尤其易于構(gòu)建抗體融合蛋白等優(yōu)越性。 結(jié)合現(xiàn)在腫瘤治療個(gè)性化概念的出現(xiàn),我們期望構(gòu)建出個(gè)體病人特有的單鏈抗體庫(kù),并以此為基礎(chǔ)篩選到針對(duì)自身腫瘤的抗體。 以此為出發(fā)點(diǎn),我們開(kāi)展了單鏈抗體的研究,從首先通過(guò)抽取健康個(gè)體的外周血并得到個(gè)體的cDNA,作為基因擴(kuò)增的模板,并參閱了之前大量的報(bào)道與抗體數(shù)據(jù)庫(kù),設(shè)計(jì)出涵蓋人體所有抗體基因的引物。利用這些引物隨機(jī)組合并進(jìn)行PCR擴(kuò)增,擴(kuò)增出的重鏈與輕鏈片段,產(chǎn)生的scFV基因型非常豐富,其中也會(huì)包含一部分在機(jī)體正常情況下被免疫耐受等過(guò)程而失去功能的抗體。就此而言,單鏈抗體(scFV)產(chǎn)生的抗體類型較傳統(tǒng)的抗體類型更加豐富,且易于基因操作。 共設(shè)計(jì)了31條涵蓋重鏈與輕鏈基因的引物,并進(jìn)行了75個(gè)獨(dú)立的PCR反應(yīng)。并將得到的重鏈與輕鏈片段以一個(gè)(GGGGS)3的彈性linker連接到一起。同時(shí),我們利用電轉(zhuǎn)的方法來(lái)構(gòu)建得到scFV單鏈抗體庫(kù),通過(guò)提高電轉(zhuǎn)的效率來(lái)構(gòu)建出更大的scFV單鏈抗體庫(kù)。,最終,我們得到了2×106的scFV單鏈抗體庫(kù)。 構(gòu)建的O型血個(gè)體的單鏈抗體用于篩選抗A,B血型的單鏈抗體,篩選是利用ELISA的方法,因此我們前期對(duì)紅細(xì)胞膜的處理及ELSA實(shí)驗(yàn)中的包被條件進(jìn)行了大量的實(shí)驗(yàn)摸索,期望得到高親和力抗原包被的條件對(duì)包被條件通過(guò)酶標(biāo)儀進(jìn)行檢測(cè),結(jié)果證實(shí)細(xì)胞膜包被條件成功,可用于ELISA篩選。
[Abstract]:Single chain antibody (Single chain FV, scFV) is one of the most widely used recombination antibodies of small molecules. It is composed of a segment of elastic link chain (linker) that connects the heavy chain variable region VH with the light chain variable region VL. The smallest functional structural unit with the full antigen-binding specificity of the parental antibody, the size of which is 1/6 of the complete antibody. It has the advantages of small molecule, strong penetration, short half-life in vivo and low immunogenicity. It can be expressed in large quantities through inclusion bodies, easy to operate genes, and especially easy to construct antibody fusion protein. With the emergence of the concept of individualized tumor therapy, we hope to construct a single chain antibody library that is specific to individual patients and screen antibodies against autotumours on the basis of this library. From this point of view, we carried out the study of scFv. First, we extracted the peripheral blood of healthy individuals and obtained individual cDNA, as template for gene amplification. We also read a large number of previous reports and antibody databases. Primers covering all human antibody genes were designed. By random combination of these primers and PCR amplification, the heavy and light chain fragments were amplified, resulting in a very rich scFV genotypes, which also contained some antibodies which were incapacitated by immune tolerance and other processes under normal conditions. In this case, the type of antibody produced by scFv (scFV) is more abundant than that of traditional antibody type, and it is easy to operate gene. A total of 31 primers covering heavy and light chain genes were designed and 75 independent PCR reactions were performed. The obtained heavy chain and light chain fragment are connected together with an elastic linker of (GGGGS) 3. At the same time, we use electroporation to construct scFV single chain antibody library. By improving the efficiency of electroporation, we construct a larger scFV single chain antibody library. Finally, we get 2 脳 10 6 scFV single chain antibody library. The single chain antibodies of O blood type individuals were used to screen the single chain antibodies against Agna B blood group. The screening method was based on ELISA. Therefore, we have done a lot of experiments on the treatment of erythrocyte membrane and the conditions of encapsulation in ELSA experiment. It was expected that the conditions of high affinity antigen coating could be detected by enzyme labeling instrument. The results showed that the conditions of membrane coating were successful and could be used for ELISA screening.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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