SEC2在莱茵衣藻中的表达及免疫学活性分析

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.

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

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 pH124 vector with Hsp70A-RBCS2 promoter and RBCS2 terminator to construct the expression vectors pH124sec2t for transformation in Chlamydomonas reinhardtii.The pH124sec2t 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-PCR analysis.Southern blot and Western blot analysis showed that sec2t gene had been integrated into the nuclear genome 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(106 / 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 experimental 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+ significantly,but it decreases the ratio of CD4+/CD8+ in the experimental mice.