摘要
采用RT-PCR与RACE方法扩增出北京油鸡腺苷酸琥珀酸裂解酶(ADSL)基因全长cDNA序列,亚克隆和序列分析结果表明:该基因开放阅读框长为1455个碱基,编码485个氨基酸;5′端非转录调控区具有典型管家基因的特征,在临近起始密码子-27号碱基发生C→T突变,该突变使得本来不是核呼吸因子2(NRF-2)结合位点的CTCC突变为NRF-2结合位点CTTC。将ADSL基因完整开放阅读框重组至融合表达载体pGEX-4T-1中,构建成北京油鸡ADSL基因融合表达载体pGEX-ADSL,转化大肠杆菌BL21(DE3),筛选阳性克隆,IPTG诱导表达。经SDS-PAGE电泳显示重组融合蛋白在约80.5kD处有特异蛋白条带出现,与预期分子量大小一致,等电点为6.79。该蛋白的表达量随诱导时间的延长而增加,5h达最高值,达到细胞总蛋白的26.9%,且主要以不可溶的包涵体形式存在,经优化表达条件,成功地获得了可溶性的融合蛋白,经Glutathione Sepharase4B凝胶纯化后Western blotting检测表明其为北京油鸡ADSL蛋白,为其进一步的生物学功能及其应用研究鉴定基础。
The full-length of the eDNA sequence of Beijing fatty chicken ADSL gene was investigated by RT-PCR and RACE in this study. The results demonstrated that the complete ADSL eDNA revealed an open reading frame of 1455 nucleotides coding for 485 amino acid residues. The promoter of chicken ADSL eDNA showed typical features of house-keeping genes： no canonical TATA and CAAT boxes, 72.65% GC in 234bp near the start codon, and there was a C-27T mutation, which caused the site CTCC mutating to the NRF-2 binding site CTTC. The complete open reading frame of the ADSL gene was inserted into expression vector pGEX-4T-1 and the fusion expression vector pGEX-ADSL was constructed. The pGEX-ADSL was transformed into Escherichia coli BL21 （DE3）, positive cloning screened, induced and expresses by IPTCt The SDS-PAGE electrophoresis results showed that there were specific bands, about 83.5 kD and an isoelectric point of 6.79, which reached 26.9% of total cell proteins induced in 5hrs, and was an insoluble inclusion body. Under optimal conditions, ADSL was purified by Glutathione Sepharase 4B affinity chromatography, and Western blotting analysis showed that the fused protein was ADSL. This research was a foundation for further research into ADSL＇s biological function and its application identification.
基金
国家"863"计划项目资助(2006AA10Z198
2007AA10Z170)
关键词
北京油鸡
腺苷酸琥珀酸裂解酶
结构分析
基因表达
Beijing fatty chicken
Adenylosuccinate lyase
Structure analysis
Gene expression
