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  本文關(guān)鍵詞：抗hTNF-α單克隆抗體的制備及其抗體基因克隆研究　出處：《中國協(xié)和醫(yī)科大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腫瘤壞死因子(TNF-α) hTNF-α抑制劑 自身免疫性疾病

【摘要】： 腫瘤壞死因子a(Tumor necrosis factor a, TNFa)是一種由活化的單核/巨噬細(xì)胞分泌的多功能細(xì)胞因子,研究表明：適量的TNFa可以協(xié)助宿主抵抗微生物入侵、殺傷某些腫瘤細(xì)胞或使體內(nèi)腫瘤組織發(fā)生出血壞死,但過量的TNFa與其它炎性因子一起產(chǎn)生多種病理性損傷,可以引起組織損傷、重癥感染性休克、自身免疫性疾病等。可以說TNF-a作為一個調(diào)節(jié)生理平衡的關(guān)鍵細(xì)胞因子,在人類對抗腫瘤及其他疾病中扮演了“雙刃劍”的角色。 通過抑制TNFa的活性作用可以對與TNFa有關(guān)的疾病產(chǎn)生治療作用,目前主要用于類風(fēng)濕關(guān)節(jié)炎、抑制腫瘤的發(fā)生、生長和轉(zhuǎn)移以及對多藥耐藥的治療。以TNFa為靶點開發(fā)的藥物包括有很多,這些藥物從不同層次不同水平抑制TNFa的產(chǎn)生和發(fā)生作用從而達(dá)到抗TNFa治療的目的。臨床研究表明抗TNFa治療的藥物是安全的。 本實驗在我室原有抗hTNF-a表達(dá)載體的基礎(chǔ)上,表達(dá)出可溶性hTNFa蛋白,之后用經(jīng)典的細(xì)胞融合的方法獲得抗hTNFa的雜交瘤細(xì)胞株；采用硫酸銨沉淀和蛋白G親和層析法純化腹水型單抗；采用ELISA酶鏈免疫分析方法測定其效價、抗體亞型和親和力；采用MTT法測定抗體阻斷hTNFa對L929細(xì)胞的細(xì)胞毒的作用和篩選拮抗型抗hTNFa單抗；流式細(xì)胞儀法測定抗體阻斷hTNFa誘導(dǎo)L929細(xì)胞凋亡的作用。最終獲得一株能穩(wěn)定分泌抗hTNFa抗體的雜交瘤細(xì)胞株XB10,分泌的抗體亞型為IgG2；該抗體能高親和力與hTNFa結(jié)合,特異性阻斷hTNFa對L929細(xì)胞的細(xì)胞毒作用和抑制細(xì)胞凋亡的發(fā)生,并呈一定的劑量依賴性。用RT-PCR和PCR方法克隆出抗hTNFa的輕、重鏈基因,與T載體連接,測序結(jié)果與PDB庫中抗體特征框架氨基酸序列相符合,是抗體的輕、重鏈基因。該研究為今后研制臨床治療型抗hTNFa的基因工程抗體奠定了實驗基礎(chǔ)。
[Abstract]:Tumor necrosis factor Tumor necrosis factor a (TNFa) is a multifunctional cytokine secreted by activated monocytes / macrophages. Studies have shown that appropriate amount of TNFa can help the host resist microbial invasion, kill some tumor cells or cause bleeding and necrosis of tumor tissue in vivo. However, excessive TNFa and other inflammatory factors together produce a variety of pathological damage, can cause tissue damage, severe septic shock. It can be said that TNF-a, as a key cytokine in regulating physiological balance, plays a "double-edged sword" role in human anti-tumor and other diseases. By inhibiting the activity of TNFa, it can be used to treat TNFa related diseases. At present, it is mainly used in rheumatoid arthritis and tumorigenesis. Growth and metastasis as well as treatment of multidrug resistance. A wide range of drugs have been developed targeting TNFa. These drugs inhibit the production and effect of TNFa from different levels and levels to achieve the purpose of anti-#en1# therapy. Clinical studies have shown that anti- TNFa drugs are safe. In this experiment, soluble hTNFa protein was expressed on the basis of the original anti-#en0# expression vector in our laboratory, and then the hybridoma cell line with anti-#en2# was obtained by classical cell fusion method. The ascites monoclonal antibody was purified by ammonium sulfate precipitation and protein G affinity chromatography. The titer, antibody subtype and affinity were determined by ELISA immunoassay. The cytotoxicity of hTNFa to L929 cells was blocked by MTT assay and the antagonistic anti hTNFa monoclonal antibody was screened. Flow cytometry was used to determine the blocking effect of hTNFa on apoptosis of L929 cells. Finally, a hybridoma cell line XB10, which could stably secrete anti-#en1# antibody, was obtained. The antibody subtype secreted was IgG2; The antibody can bind to hTNFa with high affinity and specifically block the cytotoxicity of hTNFa to L929 cells and inhibit the apoptosis of L929 cells. In a dose-dependent manner, the light and heavy chain genes of anti-#en2# were cloned by RT-PCR and PCR, and ligated with T vector. The results of sequencing are consistent with the amino acid sequence of the characteristic frame of antibody in PDB library and are light and heavy chain genes of antibody. This study will lay an experimental foundation for the development of clinical therapeutic antibody against hTNFa in the future.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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本文編號：1387450