SEC2在莱茵衣藻中的表达及免疫学活性分析  

Expression and immunological activity of enterotoxin C2 from Staphylococcus aureus in Chlamydomonas reinhardti

作　　者：李建成[1,3] 彭世清[3] 胡章立[2] 

机构地区：[1]海南大学农学院,海口570228 [2]深圳大学生命科学学院,深圳市海洋生物资源与生态环境重点实验室,深圳518060 [3]中国热带农业科学院热带生物技术研究所,农业部热带作物生物技术重点开放实验室,海口571101

出　　处：《深圳大学学报（理工版）》2012年第2期159-164,共6页Journal of Shenzhen University(Science and Engineering)

基　　金：国家自然科学基金资助项目(31070323);广东省科技计划资助项目(2009B020307013)~~

摘　　要：通过将金黄色葡萄球菌(Staphylococcus aureus,S.aureus)肠毒素C2(staphylococcal entero-toxin C2,SEC2)基因进行定点突变,获得具有超抗原性的减毒C2基因序列sec2t,把该基因插入含Hsp 70A-RBCS2启动子和RBCS2终止子的pH124载体上,构建莱茵衣藻表达载体pH124sec2t,采用'珠磨法'将该载体转入细胞壁缺陷型莱茵衣藻(Chlamydomonas reinhardtii)CC-849中,经含10μg/mL Zeomy-cin的抗性平板筛选、PCR及RT-PCR分析,获得转基因莱茵衣藻Tran-sec2t.Southern blot和Western blot分析表明,sec2t基因已整合入莱茵衣藻核基因组中,且能表达相对分子质量为26 kDa(1 Da=1 u)的目的蛋白.分别以CC-849和Tran-sec2t两种藻细胞培养液(每毫升106个细胞)喂食Balb/c小鼠,并用流式细胞仪检测分析小鼠CD3+、CD4+和CD8+细胞,结果发现,可表达减毒超抗原SEC2T的转基因莱茵衣藻Tran-sec2t能刺激小鼠T淋巴细胞的产生;与对照相比,CD4+与CD8+细胞数目比值有所降低,但CD3+和CD8+细胞有明显提高.研究结果有助于利用转基因藻生产抗肿瘤制剂和动物免疫增强剂.A superantigenic gene （sec2t） of the attenuated enterotoxin C2 was obtained by the site-directed mutagenesis of enterotoxin C2 in Staphylococcus aureus. The sec2t was inserted into the pill24 vector with Hsp 70A-RBCS2 promoter and RBCS2 terminator to construct the expression vectors pill24 sec2t for transformation in Chlamydomonas reinhardtii. The pill24 sec2t vectors were transformed into the algal cell by the glass-bead method in Chlamydomonas reinhardtii CC-849. A number of transformants appeared in the TAP plates with 10 μg/mL of Zeomycin in 3 ~ 4 weeks. The transgenic strain Tran-sec2t was further screened by PCR and RT-PCRanalysis. Southern blot and Western blot analysis showed that sec2t gene had been integrated into the nuclear ge- nome of Tran-sec2t, and a 26 kDa（ 1 Da = 1 u） SEC22T protein had been expressed in the Tran-sec2t. In order to detect the immunological activity of Tran-sec2t, the saline （0. 9% ） and cultures （10s/mL） of CC-849 and Tran- sec2t were used to feed the three groups of Balb/c mice respectively. The CD3 ＋ , CD4 ＋ and CD8 ＋ of experimen- tal mice were detected by flow cytometry. The results show that T lymphocytes proliferation of mice is stimulated by the SEC2T expressed in Tran-sec2t. To compare with T-cell response of CC-849, the Tran-sec2t leads to the increase of CD3 ＋ and CD8 ＋ siGnificantlv, but it decreases the. ratio of CD4＋/CDR ＋ in experimental mice.

关 键 词：基因工程 金黄色葡萄球菌 肠毒素C2 莱茵衣藻 超抗原 转基因藻 抗肿瘤 定点突变 

分 类 号：Q37[生物学—遗传学] R392.1[医药卫生—免疫学]