【作者】 张崇旭；

【导师】 熊正英；

【作者基本信息】 陕西师范大学 ， 运动人体科学， 2004， 硕士

【摘要】 自从1954年发现肠毒素B（SEB）以来，人们对它的研究就一直没有停止，金葡菌肠毒素B可以引起食物中毒，中毒发病较急，导致动物和人呕吐，研究表明，引起恒河猴子呕吐作用是感觉刺激经过迷走神经及交感神经至呕吐中枢。肠毒素B还可引起人和动物腹泻，人们认为肠毒素诱发腹泻至少可归因于抑制了肠腔中水分的吸收或增加了经粘膜流入肠液中的水分或是二者共同作用的结果。肠毒素B可引起伪膜性小肠炎，大肠炎而使病人死亡。一般是继腹泻之后出现休克循环衰竭，尸检可见到伴有肺泡血管的显著充血，出血性肺水肿和肝白细胞浸润。近几年的研究发现SEB是一种超抗原（superantigen，SAg），是一组具有强大刺激能力的细菌外毒素，其无需抗原提呈细胞（antigen presenting cells APC）内处理加工,能直接与APC膜上的MHC-Ⅱ类分于抗原结合槽外侧非限制性结合，以完整的蛋白质分于形式被提呈给T细胞，只需极低浓度（1-10ng/ml）即可诱发最大的应答效应，导致带有特异性T细胞受体B链可变区（T cell receptor beta chainvariable regions，TCR V_β）序列的T细胞大量活化增殖并释放出大量多种细胞因子，产生生物学效应，鉴于SAg具有特殊的生物学效应，有关SAg在抗肿瘤方面的研究日益引起了人们的注意，随着对治疗恶性肿瘤药物研究的进展，肠毒素B逐渐成为人们注意的焦点。肠毒素B作为超抗原，激活T淋巴细胞，致使其释放细胞因子，从而达到杀伤肿瘤细胞的目的。不同细胞周期的T细胞对作为超抗原的肠毒素B的反应不同。SEB是目前研究的最多的一种超抗原，最早获得的葡萄糖球菌肠毒素超抗原是从金葡菌的发酵液中提取的。首先进行产毒培养，然后进行分离纯化，但天然毒素的产量低（20-200mg/L），给后续的毒素纯化带来极大困难，本研究将SEB基因克隆进质粒P^TIG-TRX与硫氧还蛋白共表达，以可溶性表达为目的，结果在大肠杆菌BL21（DE3）中获得了良好的表达，并对表达的重组蛋白进行了纯化和免疫原性鉴定，纯化产量达150mg/l，可用于中试。目前对SEB中毒治疗主要是一些传统疗法，包括输液，换气以及肾透析等。国外一些研究者以SEs（SEA,SEB，SEC,SED,SEE）共有的保守氨基酸为靶，研制抗SEs的抗体，其动物实验取得了成功，但未见进一步报道。单链抗体分子量小，免疫原性低半衰期短，在血液循环中清除快，可以作为SEB食物中毒的特效药以及快速检测。噬菌体表面呈现技术（Phage display technology）是利用分子生物学方法将外源多肽、蛋白、抗体等融合并呈现于噬菌体表面，构成表型与基因型直接相连的随机多肤、蛋白、抗体库。这种外源多肤、蛋白、抗体可以识别相应的结合分子而被筛选出来。由于其技术上的优越性;易于扩增和选择;筛选得到的氨基酸序列可以通过DNA测序确定。因而得到广泛应用和迅速发展。本研究用本研究主要通过PCR，克隆SEB基因到表达载体PTIG一Trx中，引入大肠杆菌BLZI中，诱导表达，利用sDs一PAGE，westem检测和鉴定.表达出的SEB，经纯化后的SEB免疫兔子，取脾，提取总RNA，做RT一PCR，以。DAN为摸板，扩增出vH，VL片段，用柔性linker连接后，连接到噬菌粒PCANTAB，E中，建立抗体库，本研究通过改构的噬菌粒成功的构建了单链抗体库，为筛库和SEB中毒检测，治疗等奠定了基础。结果获得了良好的可溶性表达，纯化产量为150mg几。纯化产物免疫兔子后，经RT一PCR和三轮PCR成功的构建了单链抗体库，库容达10’‘，可用于下一步工作。更多还原

【Abstract】 The study of staphylococcal enterotoxin B (SEB ) have never been stopped since it was discovered in 1954, SEB is a common cause of food poisoning and toxic shock. SEB can make Rhesus monkeys vomit, this mechanism is relate to stimulation of vomit nerve centre of pneumogastric nerve and sympathetic nerve,it also can conduce diarrhea,excessive and frequent evacuation of watery feces, usually indicating gastrointestinal distress or disorder,even shock and death. SEB is also an important member of the superantigen family, which exerts a number of pathological effects in the human, as well as susceptible animals. With the development of anti-tumor medicines, more attention has focused on it. As a superantigen, SEB possesses the ability to bind to major histocompatibility complex class II molecules and be recognized by T cells bearing certain T-cell receptor (TCR) V beta alleles. Superantigen-mediated T cell activation requires the participation of antigen-presenting cells (APC). Once superantigen has bound class II MHC molecules on the surface of APC, it then can interact with the T cell receptor to induce T cell activation. Superantigen-mediated T lymphocyte activation, along with its consequent cytokine production is thought to be the basis for the pathophysiology of conditions such as toxic shock syndrome and possibly rheumatoid arthritis. SEB can activate T-lymphocytes, include the release of lymphokine and cause the lysis of tumor cells. The output of SEB from native staphylococcal is low and it is hard to purify. In this study, we want to clone SEB gene and express soluble SEB in E.coli. At this time there are no vaccines or therapeutics to protect against SEB exposure. We also want to construct a single-chain fragment (ScFv) library in which in order to select antibody binding specifically to SEB. The gene of SEB is amplifyied with PCR from SEB producing strain 1169 and is cloned into expression vector pTIG-Trx for soluble express in E.coli.The crude expressed protein was identified by SDS-PAGE and Western, then the product was purified by Probond resin. We use the pure SEB immunize rabbit,extact total RNA from spleen, by means of phage display technology we construct combinatorial immunoglobulin library. The gene of SEB is obtained by PCR, and get soluable expression in the host strain BL21 (DE3) , the last purifyied production is about 150mg/l. By means of phage display we construct a library, capacity of this library is about 1011, it can be used in the next processs.更多还原