【摘要】： 自1986年美國FDA正式批準(zhǔn)抗CD3單抗作為臨床上治療腎移植排斥反應(yīng)的新藥上市以來,抗CD3單抗以其具有激活和抑制人T細(xì)胞雙向功能的特點,在各種器官移植排斥反應(yīng)、腫瘤及自身免疫性疾病治療中顯示出其廣闊的應(yīng)用前景。但目前臨床上應(yīng)用的CD3單抗主要是鼠源性的,鼠源單抗在人體內(nèi)應(yīng)用時產(chǎn)生排斥反應(yīng)、半衰期短、活性下降、異性蛋白反應(yīng)等問題,嚴(yán)重限制其在臨床治療中的廣泛應(yīng)用。因此,近年來對于人源性的抗CD3抗體的研究成為熱點。大多數(shù)抗CD3抗體基因構(gòu)建后主要選用大腸桿菌和哺乳動物細(xì)胞為表達體系,這些表達體系都存在許多缺點。 酵母表達體系具備易于操作,遺傳背景清楚,生產(chǎn)成本低等原核生物的特點,又具有真核生物的折疊、分泌機制,可以完成真核生物特異的蛋白酶解、折疊、糖基化等翻譯后修飾過程,且生長速度快,培養(yǎng)成分簡單,能適應(yīng)工業(yè)化生產(chǎn)的需要。本研究選擇酵母偏愛密碼子同義替換鼠源性抗CD3重鏈可變區(qū)基因(Vh),與人源性重鏈恒定區(qū)(Ch)基因連接,構(gòu)建完整的人鼠嵌合抗體。將構(gòu)建的完整的人鼠嵌合表達載體轉(zhuǎn)化X-33畢赤酵母,建立兼具原核系統(tǒng)良好操作性和真核系統(tǒng)后加工性的重組抗CD3畢赤酵母真核表達體系。通過ELISA和MTT的鑒定,篩選出高表達重組抗CD3的酵母菌株。結(jié)果表明,本研究正確構(gòu)建了重組抗CD3抗體畢赤酵母真核表達體系,經(jīng)甲醇誘導(dǎo)能夠分泌具有活性的重組抗CD3抗體蛋白,相對分子量為55kD;重組抗CD3抗體的表達具有累積效應(yīng),產(chǎn)量在一段時間內(nèi)隨表達時間延長而增加。本研究為進一步用畢赤酵母作為生物反應(yīng)器大規(guī)模生產(chǎn)抗CD3抗體提供了實驗依據(jù)。
[Abstract]:Since the United States FDA officially approved anti-CD3 monoclonal antibody as a new drug for the treatment of renal transplantation rejection in 1986, anti-CD3 monoclonal antibody has been used in various organ transplantation rejection because of its bidirectional function of activating and inhibiting human T cells. The treatment of tumor and autoimmune disease shows its broad application prospect. However, the current clinical use of CD3 monoclonal antibody is mainly murine origin, the mouse source monoclonal antibody in human body produced rejection, short half-life, decreased activity, heterosexual protein reaction and other problems, which seriously limits its wide application in clinical treatment. Therefore, the study of human anti-CD3 antibody has become a hot topic in recent years. Escherichia coli and mammalian cells were used as expression systems after construction of anti CD3 antibody genes, and these expression systems had many disadvantages. The yeast expression system is characterized by easy operation, clear genetic background, low production cost and the mechanism of eukaryote folding and secretion, which can complete the proteolysis and folding of eukaryote. Glycosylation and other post-translational modification processes, with rapid growth, simple culture ingredients, can meet the needs of industrial production. In this study, yeast preference codon synonymous substitution of mouse anti CD3 heavy chain variable region gene (Vh), and human origin heavy chain constant region (Ch) gene was selected to construct a complete human mouse chimeric antibody. The constructed human mouse chimeric expression vector was transformed into Pichia pastoris X-33, and a recombinant eukaryotic expression system of anti-Pichia pastoris CD3 was established with good prokaryotic system operation and post-processing of eukaryotic system. Through the identification of ELISA and MTT, yeast strains with high expression of recombinant CD3 resistant to CD3 were screened out. The results showed that the eukaryotic expression system of recombinant anti CD3 antibody Pichia pastoris was constructed correctly. The recombinant anti CD3 antibody protein was secreted by methanol and its relative molecular weight was 55 kD. The expression of recombinant anti-CD3 antibody had cumulative effect, and the yield increased with the time of expression. This study provides experimental basis for mass production of anti-CD3 antibodies using Pichia pastoris as a bioreactor.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：Q786;R392

【參考文獻】

相關(guān)期刊論文 前3條

1 楊凡,施偉民,陸震宇,孫向華,于德華;抗CD3單抗促進腫瘤細(xì)胞凋亡的研究[J];上海醫(yī)學(xué);2003年S1期

2 熊冬生,許元富,楊純正,彭暉,邵曉楓,劉漢芝,韓俊嶺,賴增祖,朱禎平;抗CD3/抗CD20雙特異雙鏈抗體的生物學(xué)活性研究[J];中華微生物學(xué)和免疫學(xué)雜志;2001年06期

3 王琰;基因工程抗體研究進展[J];中國免疫學(xué)雜志;1999年05期

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本文編號：2391965