| 摘要: |
| 根据克隆得到的中国对虾(Fenneropenaeus chinensis)CYP4 基因开放阅读框设计引物, 构建了中国对虾CYP4 基因原核表达载体p28a - CYP4, 并对重组菌株Rosetta/28a-CYP4 的原核表达条件进行了优化。结果表明: p28a-CYP4 转化Rosetta 后可实现CYP4 基因的原核表达, SDS-PAGE 分析显示其在56.0kDa 处有显著诱导条带; 通过对诱导温度、IPTG 浓度、诱导时机(OD600)及诱导时间的优化表明, 重组菌株Rosetta/p28a-CYP4 的最佳诱导温度为37℃, 最佳IPTG 浓度为1.2 mmol/L, 最佳诱导时机及诱导时间分别为0.59 和6 h。 |
| 关键词: 中国对虾(Fenneropenaeus chinensis) 细胞色素P450 原核表达 |
| DOI： |
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| 基金项目:公益性行业专项(200803012); 虾产业技术体系(nycytx-46); 山东省博士后创新基金(200803024) |
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| Optimization of prokaryotic expression of the CYP4 gene of Chinese shrimp (Fenneropenaeus chinensis) |
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| Abstract: |
| Specific primers were designed according to the open reading frame (ORF) sequence of CYP4 cDNA of Chinese shrimp (Fenneropenaeus chinensis). Prokaryotic expression vector p28a-CYP4 was constructed and the prokaryotic expression conditions were optimized. The results showed that the Rosetta strain after being transformed with the recombinant expression vector accumulated high amount of recombinant protein. SDS-PAGE analysis revealed that the recombinant protein was about 56.0 kDa. The recombinant strain Rosetta/p28a-CYP4 was induced at different temperatures, IPTG concentrations, OD600, and different times. The recombinant accumulated highest amount of recombinant protein after IPTG (1.2mmol/L) induction (OD600=0.59) at 37℃ for 6 h. |
| Key words: Chinese shrimp (Fenneropenaeus chinensis) cytochrome P450 prokaryotic expression |
