金黄色葡萄球菌肠毒素C2突变体构建及其生物活性研究

发布时间：2018-01-27 15:52

本文关键词： 超抗原金黄色葡萄球菌肠毒素C2 点突变 PBMC增殖体外抑瘤　出处：《辽宁大学》2007年硕士论文　论文类型：学位论文

【摘要】： 金黄色葡萄球菌肠毒素(Staphylococcal enterotoxins, SEs)是一类具有高度生物学活性的蛋白质、能活化T细胞的超抗原。与普通抗原不同,超抗原无须经抗原提呈细胞(Antigen presenting cell, APC)加工处理,可以完整的蛋白分子形式直接与抗原提呈细胞膜上的MHC-II(Major histocompatibility complex class II molecules, MHC-II)类分子及T细胞受体Vβ区(Variable pacts of the T cells receptor, TCR V?)特异性结合,刺激T细胞增殖,产生大量有生物学活性的细胞因子和药性效应,引发超级抗原依赖的细胞介导的细胞毒性作用(Superantigen dependent cell-mediated cytotoxicity, SDCC)和其它免疫效应。利用SEs的这一特点,沈阳协和集团开发出以金黄色葡萄球菌肠毒素C2(SEC2)为主要成分的新型抗肿瘤生物制剂——“高聚生”,临床用于治疗肺癌、食道癌、卵巢癌、晚期肝癌、大肠癌、血癌、膀胱癌等恶性肿瘤,效果显著。但作为潜在的肠胃毒素,SEC2可引起食物中毒,引发腹绞痛和严重腹泻,这严重影响和限制了其在临床的广泛应用。肠毒素结构与其致病性及致病能力关系密切,肠毒素所引发的催吐效应和T细胞诱导效应无关,而与胱氨酸环(Cys93, Cys110)及His118有关。为此,本研究利用基因定点突变技术,选择性替换SEC2中110位Cys和118位His,获得了相应的突变基因,并在大肠杆菌中实现高效表达。体外PBMC(Peripheral blood mononuclear cell, PBMC)增殖和抑瘤实验被用于分析突变体蛋白的超抗原活性和抑瘤活性。本研究结果如下: 1)利用PCR定点突变技术,SEC2中110位Cys成功的被Ala和Ser替换,118位的His被Tyr替换。带有突变基因的表达质粒在E. coli BL21 (DE3)中经IPTG诱导,实现高效表达。SDS-PAGE电泳结果表明,经Ni-NTA亲和层析柱和Sephadex G-75纯化获得的突变体蛋白呈单一条带; 2) PBMC增殖研究表明,所有突变体蛋白均能有效刺激PBMC增殖,与SEC2无明显差异,在200 ng/ml时,增殖能力最强。 3)体外抑瘤实验表明,所有突变体都在不同程度上抑制了大肠癌细胞CX-1的生长,其中SEC2(C93S, C110S)抑瘤效果最为明显,在2 ng/ml和500 ng/ml之间,抑瘤效果均略高于SEC2。综上所述,本研究成功的利用基因定点突变技术,替换了SEC2中与催吐有关的氨基酸,获得了超抗原活性和抑瘤活性基本保持不变的SEC2突变体,从而为开发无毒、无副作用的新型抗肿瘤生物制剂奠定了工作基础。
[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.
【学位授予单位】：辽宁大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R378

【引证文献】