[1]李建成,彭世清,胡章立.SEC2在莱茵衣藻中的表达及免疫学活性分析[J].深圳大学学报理工版,2012,29(No.2(095-188)):159-164.[doi:10.3724/SP.J.1249.2012.02159]
　LI Jian-cheng,PENG Shi-qing,et al.Expression and immunological activity of enterotoxin C2 from Staphylococcus aureus in Chlamydomonas reinhardti[J].Journal of Shenzhen University Science and Engineering,2012,29(No.2(095-188)):159-164.[doi:10.3724/SP.J.1249.2012.02159]
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SEC2在莱茵衣藻中的表达及免疫学活性分析()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第29卷
期数:
2012年No.2(095-188)
页码:
159-164
栏目:
生物工程
出版日期:
2012-03-23

文章信息/Info

Title:
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
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
Author(s):
LI Jian-cheng1;  3;  PENG Shi-qing3;  and HU Zhang-li2
1) College of Agriculture, Hainan University, Haikou 570228, P.R.China
2) Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Science, Shenzhen University, Shenzhen 518060, P.R.China
3) Ministry of Agriculture Key Biotechnology Laboratory for Tropical Crops, The Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, P.R.China
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
Keywords:
genetic engineering;  Staphylococcus aureus;  enterotoxin C2;  Chlamydomonas reinhardtii;  superantigen;  transgenic algae;  anti-tumor;  site-directed mutagenesis
分类号:
Q 37；R 392.1
DOI:
10.3724/SP.J.1249.2012.02159
文献标志码:
A
摘要:
通过将金黄色葡萄球菌（Staphylococcus aureus, S.aureus）肠毒素C2(staphylococcal enterotoxin 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Hsp70A-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10 μg/mL Zeomycin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．
Abstract:
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.

参考文献/References:

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备注/Memo

备注/Memo:
Received：2012-01-04；Accepted：2012-02-14
Foundation：NationalNaturalScienceFoundationofChina(31070323),ScienceandTechnologyPlanningProjectofGuangdongProvince(2009B020307013)
Corresponding author：Professor HU Zhang-li. E-mail: huzl@szu.edu.cn
Citation：LI Jian-cheng, PENG Shi-qing, HU Zhang-li. Expression and immunological activity of enterotoxin C2 from Staphylococcus aureus in Chlamydomonas reinhardtii［J］. Journal of Shenzhen University Science and Engineering, 2012, 29(2): 159-164.（in Chinese）
基金项目：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)
作者简介：李建成(1985-)，男 (汉族)，湖南省常德市人，海南大学硕士研究生. E-mail: lijc1007@163.com
引文：李建成，彭世清，胡章立. SEC2在莱茵衣藻中表达及免疫学活性分析［J］. 深圳大学学报理工版，2012，29（2）：159-164.
更新日期/Last Update: 2012-03-28