本文關(guān)鍵詞： 超抗原 金黃色葡萄球菌腸毒素C2 點(diǎn)突變 PBMC增殖 體外抑瘤　出處：《遼寧大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 金黃色葡萄球菌腸毒素(Staphylococcal enterotoxins, SEs)是一類具有高度生物學(xué)活性的蛋白質(zhì)、能活化T細(xì)胞的超抗原。與普通抗原不同,超抗原無須經(jīng)抗原提呈細(xì)胞(Antigen presenting cell, APC)加工處理,可以完整的蛋白分子形式直接與抗原提呈細(xì)胞膜上的MHC-II(Major histocompatibility complex class II molecules, MHC-II)類分子及T細(xì)胞受體Vβ區(qū)(Variable pacts of the T cells receptor, TCR V?)特異性結(jié)合,刺激T細(xì)胞增殖,產(chǎn)生大量有生物學(xué)活性的細(xì)胞因子和藥性效應(yīng),引發(fā)超級抗原依賴的細(xì)胞介導(dǎo)的細(xì)胞毒性作用(Superantigen dependent cell-mediated cytotoxicity, SDCC)和其它免疫效應(yīng)。利用SEs的這一特點(diǎn),沈陽協(xié)和集團(tuán)開發(fā)出以金黃色葡萄球菌腸毒素C2(SEC2)為主要成分的新型抗腫瘤生物制劑——“高聚生”,臨床用于治療肺癌、食道癌、卵巢癌、晚期肝癌、大腸癌、血癌、膀胱癌等惡性腫瘤,效果顯著。但作為潛在的腸胃毒素,SEC2可引起食物中毒,引發(fā)腹絞痛和嚴(yán)重腹瀉,這嚴(yán)重影響和限制了其在臨床的廣泛應(yīng)用。 腸毒素結(jié)構(gòu)與其致病性及致病能力關(guān)系密切,腸毒素所引發(fā)的催吐效應(yīng)和T細(xì)胞誘導(dǎo)效應(yīng)無關(guān),而與胱氨酸環(huán)(Cys93, Cys110)及His118有關(guān)。為此,本研究利用基因定點(diǎn)突變技術(shù),選擇性替換SEC2中110位Cys和118位His,獲得了相應(yīng)的突變基因,并在大腸桿菌中實(shí)現(xiàn)高效表達(dá)。體外PBMC(Peripheral blood mononuclear cell, PBMC)增殖和抑瘤實(shí)驗(yàn)被用于分析突變體蛋白的超抗原活性和抑瘤活性。本研究結(jié)果如下: 1)利用PCR定點(diǎn)突變技術(shù),SEC2中110位Cys成功的被Ala和Ser替換,118位的His被Tyr替換。帶有突變基因的表達(dá)質(zhì)粒在E. coli BL21 (DE3)中經(jīng)IPTG誘導(dǎo),實(shí)現(xiàn)高效表達(dá)。SDS-PAGE電泳結(jié)果表明,經(jīng)Ni-NTA親和層析柱和Sephadex G-75純化獲得的突變體蛋白呈單一條帶; 2) PBMC增殖研究表明,所有突變體蛋白均能有效刺激PBMC增殖,與SEC2無明顯差異,在200 ng/ml時,增殖能力最強(qiáng)。 3)體外抑瘤實(shí)驗(yàn)表明,所有突變體都在不同程度上抑制了大腸癌細(xì)胞CX-1的生長,其中SEC2(C93S, C110S)抑瘤效果最為明顯,在2 ng/ml和500 ng/ml之間,抑瘤效果均略高于SEC2。 綜上所述,本研究成功的利用基因定點(diǎn)突變技術(shù),替換了SEC2中與催吐有關(guān)的氨基酸,獲得了超抗原活性和抑瘤活性基本保持不變的SEC2突變體,從而為開發(fā)無毒、無副作用的新型抗腫瘤生物制劑奠定了工作基礎(chǔ)。
[Abstract]:Staphylococcal enterotoxin (SES) is a class of proteins with high biological activity. The superantigen that can activate T cell. Unlike ordinary antigen, superantigen is processed without antigen presenting cell antigen presenting cell (APC). A complete protein molecule can be expressed directly with MHC-II on the cell membrane. Major histocompatibility complex class II molecules. MHC-II) class molecules and T cell receptor V 尾 pacts of the T cells receptor, TCR V? Specific binding stimulates the proliferation of T cells and produces a large number of bioactive cytokines and drug effects. Initiate superantigen-dependent cell-mediated cytotoxicity (. Superantigen dependent cell-mediated cytotoxicity. SDCC) and other immune effects. Take advantage of this characteristic of SEs. Shenyang Union Group has developed a new anti-tumor biological agent, "high agglutination", which is mainly composed of Staphylococcus aureus enterotoxin C2SEC2, which is used in the treatment of lung cancer, esophagus cancer and ovarian cancer. Advanced liver cancer, colorectal cancer, blood cancer, bladder cancer and other malignant tumors, the effect is significant. But as a potential gastrointestinal toxin, SEC2 can cause food poisoning, lead to abdominal colic and severe diarrhea. This seriously affects and limits its wide application in clinical practice. The structure of enterotoxin was closely related to its pathogenicity and pathogenicity. The emetic effect induced by enterotoxin was not related to T cell induction, but to cysteine cyclization Cys93. Cys110) and His118. In this study, we used site-directed mutagenesis to selectively replace 110-bit Cys and 118-bit His in SEC2. The mutant gene was obtained and expressed in E. coli. PBMC(Peripheral blood mononuclear cell was obtained in vitro. PBMC) proliferation and tumor inhibition tests were used to analyze the superantigen and tumor inhibition activities of mutant proteins. The results are as follows: 1) the 110-bit Cys in SEC2 was successfully replaced by Ala and Ser using PCR site-directed mutation technique. The 118-bit His was replaced by Tyr. The expression plasmid with mutant gene was induced by IPTG in E. coli BL21. SDS-PAGE electrophoresis showed that the mutant protein purified by Ni-NTA affinity chromatography and Sephadex G-75 showed a single band. 2) PBMC proliferation studies showed that all mutant proteins could effectively stimulate the proliferation of PBMC, and there was no significant difference between them and SEC2. At 200 ng/ml, the ability of proliferation was the strongest. 3) in vitro tumor inhibition experiments showed that all the mutants inhibited the growth of colorectal cancer cell CX-1 to varying degrees, among which SEC2C93S and C110S were the most effective. In the range of 2 ng/ml and 500 ng/ml, the inhibitory effect was slightly higher than that of SEC2. In conclusion, this study successfully used gene site-directed mutation technology to replace the amino acids related to emesis in SEC2, and obtained SEC2 mutants with the same superantigen activity and tumor inhibition activity. It lays a foundation for the development of non-toxic and non-side-effect anti-tumor biological agents.
【學(xué)位授予單位】：遼寧大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R378

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 邢皓;高雅;趙磊;;腸毒素蛋白結(jié)構(gòu)改造與其抗腫瘤力[J];中國科技信息;2012年16期

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本文編號：1468830