本文選題：CD20分子　切入點(diǎn)：抗體　出處：《聊城大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：非霍奇金淋巴瘤是一種常見的B細(xì)胞型淋巴瘤�，F(xiàn)代醫(yī)療技術(shù)中的放化療技術(shù)已經(jīng)不能滿足人類迫切治療該類疾病的渴求,而具有靶向治療機(jī)制的單克隆抗體方法開始被人們廣泛研究和應(yīng)用。研究發(fā)現(xiàn)CD20分子表達(dá)于NHL細(xì)胞表面,則此分子是靶向治療的理想靶點(diǎn)。利妥昔單抗(Rituxiamb)是第一個被美國食品藥品監(jiān)督管理局(FDA)批準(zhǔn)上市并用于治療NHL的單抗藥物。但因Rituximab分子中含有30%的鼠源序列,患者在長期用藥以后,易引起人抗鼠抗體免疫應(yīng)答(HAMA)而產(chǎn)生嚴(yán)重的不良反應(yīng)。同時,Rituximab的臨床反應(yīng)率只有50%,完全緩解率僅有10%。因此,嚴(yán)重制約了Rituximab在臨床中的長期應(yīng)用。針對這一現(xiàn)象,人們開始對Rituximab進(jìn)行改造,有的用與鼠源序列相似的人骨架區(qū)來替換Rituximab中的鼠源框架區(qū),有的用提高Rituximab的親和力來加強(qiáng)抗腫瘤活性。如將兩者同時進(jìn)行改造,則殺傷腫瘤細(xì)胞的效果有可能比只是改造其中一方面的效果要好。目的:改造新型的抗體可以從親和力和免疫原性兩方面進(jìn)行,可通過提高親和力來增加抗原與抗體的識別和結(jié)合,對于免疫原性主要是通過降低抗體中的鼠源序列來減少在人體內(nèi)的抗性。本文旨在構(gòu)建較高親和力和較低免疫原性的CD20抗體,并驗(yàn)證是否具有相應(yīng)的生物學(xué)活性。方法:1.通過專利分析,進(jìn)行高親和力和人源化的設(shè)計改造,將CD20H和CD20L分別克隆到真核表達(dá)載體pc DNA3.1(+)和pc DNA3.1/ZEO(+)中去,利用轉(zhuǎn)染試劑jet PEI將輕重鏈表達(dá)載體共轉(zhuǎn)染中國倉鼠卵巢細(xì)胞CHO-K1,通過有限稀釋法進(jìn)行單克隆細(xì)胞株的篩選,通過逆轉(zhuǎn)錄PCR檢測m RNA,ELISA檢測表達(dá)量,并將貼壁細(xì)胞馴化成懸浮培養(yǎng)。2.利用流式細(xì)胞術(shù)檢測細(xì)胞上清與CD20陽性細(xì)胞Raji的結(jié)合活性,并對其生物學(xué)功能進(jìn)行檢測,如細(xì)胞殺傷活性、ADCC效應(yīng)以及CDC效應(yīng)。結(jié)果:1.獲得了目的抗體輕鏈和重鏈的基因,構(gòu)建重組表達(dá)載體pc DNA3.1/ZEO(+)-CD20L和pc DNA3.1(+)-CD20H,可在CHO-K1細(xì)胞中穩(wěn)定表達(dá),并篩選了17株單克隆細(xì)胞株,RT-PCR證明目的基因在CHO細(xì)胞中成功的表達(dá),ELISA法測得培養(yǎng)上清的含量在0.45-4.72μg/m L之間,單抗細(xì)胞株在無血清培養(yǎng)基中能夠懸浮生長。流式檢測表明上清中的抗體可與Raji細(xì)胞結(jié)合,同時培養(yǎng)上清具有較好的ADCC與CDC殺傷活性,但直接的細(xì)胞殺傷活性的結(jié)果并不明顯。
[Abstract]:Non-Hodgkin 's lymphoma is a common type of B-cell lymphoma. However, monoclonal antibody methods with targeted therapeutic mechanism have been widely studied and applied. It has been found that CD20 molecules are expressed on the surface of NHL cells. Rituxiamb was the first drug to be approved by the U.S. Food and Drug Administration for use in the treatment of NHL. But because the Rituximab molecule contains 30% of the mouse origin sequence, After a long period of administration, patients are prone to serious adverse reactions caused by human anti-mouse antibody immune response (Hama). Meanwhile, the clinical reaction rate of Rituximab is only 50, and the complete remission rate is only 10. The long-term application of Rituximab in clinical practice has been seriously restricted. In response to this phenomenon, people began to modify Rituximab, and some replaced the rodent frame region in Rituximab with a human skeleton region similar to the murine origin sequence. Some increase the affinity of Rituximab to enhance its antitumor activity. The effect of killing tumor cells is likely to be better than that of just modifying one of them. Objective: the modification of new antibodies can be carried out both in terms of affinity and immunogenicity. The recognition and binding of antigens and antibodies can be enhanced by increasing affinity. The aim of this study is to construct CD20 antibodies with high affinity and low immunogenicity. Method: 1.Through patent analysis, the design of high affinity and humanization was modified, and CD20H and CD20L were cloned into eukaryotic expression vectors pcDNA3.1 () and pcDNA3.1 / ZEO (), respectively. Chinese hamster ovarian cell line CHO-K1 was co-transfected with jet PEI. The monoclonal cell lines were screened by limited dilution method, and the expression levels of CHO-K1 were detected by reverse transcription PCR (RT-PCR) and mRNA-ELISA. The adherent cells were domesticated into suspension culture. 2. Flow cytometry was used to detect the binding activity of the supernatant to Raji and its biological function. Results: 1. The gene of light chain and heavy chain of target antibody was obtained, and the recombinant expression vector pcDNA3.1% ZEO (pc-CD20L and pc-CD20H) was constructed, which could be expressed stably in CHO-K1 cells. In addition, 17 monoclonal cell lines were screened by RT-PCR. The successful expression of the target gene in CHO cells was determined by Elisa. The supernatant of culture was between 0.45-4.72 渭 g / mL. Flow cytometry showed that the antibody in the supernatant could bind to Raji cells, and the supernatant had better ADCC and CDC killing activity, but the results of direct cytotoxicity were not obvious.
【學(xué)位授予單位】：聊城大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 曹彩紅;趙曙;張清媛;;B細(xì)胞淋巴瘤信號通路及靶向治療的最新進(jìn)展[J];臨床血液學(xué)雜志;2015年01期

2 王雪瑩;姬菩忠;馬琦;;天祝白牦牛金屬硫蛋白基因(MT-1)全序列分子克隆及生物信息學(xué)分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報;2014年07期

3 侯麗霞;王文杰;劉新;;葡萄VvWRKY13轉(zhuǎn)錄因子的生物信息學(xué)分析[J];北方園藝;2013年13期

4 金t，

本文編號：1647502