论文标题:超抗原SEA的基因克隆、活性蛋白制备及其诱导T细胞免疫反应的研究
Cloning of Superantigen SEA, Preparation of Active Protein and the Study of the T Cells Reaction Induced by SEA
论文作者 宋宏萍
论文导师 隋延仿,论文学位 硕士,论文专业 病理学与病理生理学
论文单位 第四军医大学,点击次数 644,论文页数 76页File Size3574k
2005-05-01论文网 http://www.lw23.com/lunwen_337377/ 超抗原;葡萄球菌肠毒素A:原核表达;肿瘤免疫治疗;免疫耐受;T细胞无能
Superantigen; Staphylococcal enterotoxin A; Prokaryotic expression; Tumor immunotherapy; Immunological tolerance; T-cell anergy
超抗原是一种强大的T细胞激活剂,与普通抗原不同,超抗原无需抗原提成细胞的加工处理,即可以完整蛋白形式一端结合于MHCⅡ类分子肽结合槽的外侧,另一端结合于T细胞表面的TCR Vβ区,因此微量的超抗原便可以激活5%-20%的T细胞。超抗原的这种强大的免疫激活能力,使人们一直在尝试将超抗原应用于肿瘤免疫治疗,试图借助超抗原这一“外力”,以提高机体本身抗肿瘤免疫能力,并在实验中取得良好的效果。但在对超抗原的研究中发现,超抗原在激活T细胞后,会出现T细胞凋亡和无能现象,导致对超抗原的再刺激反应性降低。超抗原的这种能够引起免疫耐受的特点,限制了其在肿瘤治疗中的疗效。SEA是由葡萄球菌产生的一种外毒素,为目前研究最广泛的一种超抗原,也是唯一进入肿瘤治疗临床试验的超抗原。为了明确SEA体内作用后诱导T细胞反应的规律,指导SEA肿瘤治疗用药方式,以提高SEA肿瘤治疗疗效,本研究首先克隆SEA基因、构建SEA的原核表达载体,得到具有活性
Superantigens are extremely potent activators of T cells. In contrast toconventional antigens, superantigens needn"t be processed by APCs, and canbind outside the classical antigen-binding groove of major histocompatibilitycomplex (MHC) class II and Vβ chain of TCR by intact protein form. Sominim superangitens can activate a large proportion (5%-20%) of T cells.Because the strong T cells activation potency, superantigens have beeninvestigated extensively as therapeutic agents in tumor therapy. People wantto borrow the potency of superantigens to elevate the antitumorimmunoreaction of human bodies, and the results of experiments have provedthe feasibility and achieved good effects. However, although exposure ofmature T cells to superantigen generally induces a marked proliferativeresponse in vivo, this often followed by antigen-specific tolerance. Suchtolerance reflects either anergy or clonal elimination. Such property ofsuperantigen strongly limits its antitumor therapy effect. Staphylococcal entertoxin A (SEA) which excreted by staphylococcus, is the most extensively investigated superantigen and also the only one which have been used in the clinical I trials on superantigen antitumor therapy. In the study, we constraucted the prokaryotic expression vector of SEA and gained SEA protein with biological activity. The SEA polyclonal antibody was successfully prepared, and the property of T cell anergy induced by SEA was studied.1. SEA gene was cloned from the genome of FRI 100, the standard SEA production strain cells, by PCR, and its sequence was identical to the report in GeneBank. The analysis of protein encoded by SEA gene showed that the first 24aa segment was a signal peptide. The signal peptide is a helicalmembranous region with high hydrophobicity, which influences the prokaryotic expression ability very much. So the expression plasmid of 702 bp which encode SEA mature protein were constructed and expressed in E coli. The obtained protein was isolated and purified, then we got the purified SEA protein and the animal experiment showed that it had good biological activity. The rabbit was immunized with the denaturalized SEA protein, and the antiserum was obtained and purified by salting out. ELISA showed the titer of antiserum was higher than 1:1,000,000. The results of Western blot and imrnunofluorescence experiments indicated that the antiserum could specifically bind with SEA2. The in vitro SEA stimulating mice spleen lymphocytes proliferation experiment showed that in a certain range of SEA concentration, the extent of lymphocytes proliferation was dependent on the concentration of SEA. When the concentration of lymphocytes was 1 × 107/mL, 1 μg/mL was the optimum stimulating concentration. The results of peripheral blood lymphocytesmensuration after SEA intraperitoneal injection showed that mice injected with 10 μg SEA had a marked increase of their peripheral blood lymphocytes (p<0.05). The peak was around the third day after SEA injection, then the lymphocytes reduced gradually and returned to the normal level on the fourteenth day; whereas mice injected with 1 μg or 0.1 μg SEA did not show the increase of their peripheral bood lymphocytes. In the experiment of induction of T cells anergy by SEA administered in vivo, C57BL/6 mice were injected intrperitoneally with SEA, then at different day after SEA injection the spleen lymphocytes were isolated and were resitmulated with optimum concentration of SEA (1 μg/mL) in vitro. The results showed: compared with control, on the ninth and fourteenth day after SEA injection, the proliferations of mice spleen lymphocytes restimulated with SEA reduced markedly (p<0.05), however on the third day, the proliferation did not decrease, but increased (p<0.05). This kind of change in lymphocytes reactive ability with SEA restimulation was not related with the dose of SEA intraperitoneal injection, since the lymphocytes from mice injected with 10 μg, 1 ug or 0.1 ug all have showed markedly reduced proliferative capacity. When the concentration
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