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鸡Ii基因的克隆、鉴定及其原核表达
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摘要:
从ConA诱导的5周龄SPF鸡脾细胞中提取总RNA,用自行设计的一对引物通过RT—PCR方法扩增出鸡恒定链(invariant chain,Ii)基因片段。经酶切鉴定后,再将该片段克隆到pcDNA3载体中,测定其DNA序列,结果表明该片段长度为669bp,其DNA序列与GenBank登录的基本一致。再将该片段插入原核表达载体PGEX-4T-1,并导入菌株BL21,经诱导培养、蛋白提取和SDS—PAGE,获得分子量约为50kD的特定蛋白条带,表明所克隆的鸡Ii基因在原核细胞中得到表达。
关键词:  鸡 Ii基因 克隆 原核表达
DOI:
修订日期:2003-07-25
基金项目:国家自然科学基金;30270974;
Cloning and Identifying of Chicken Invariant Chain Gene and Its Expression in Prokaryotic Cells
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Abstract:
A total RNA was obtained from 5 weeks old SPF chicken spleen cells stimulated by ConA, then cDNA of invariant chain (Ii) gene was cloned from the RNA by reverse transcription (RT)-PCR with a pair of designed primers.The length of this cloned fragment was 669 bp and was identified by a restriction endonuclease cleavage.For sequencing,it was further inserted into plasmid pcDNA3,and showed identical with the chicken Ii gene registered in the GenBank. This fragment was inserted into PGEX-4T-1, a prokaryotic expressive plasmid, and the latter was transferred into the strain BL21. After the bacteria were induced and cultured, expressed protein was separated. A band with 50 000 of molecular weight was observed on SDS-PAGE. All these results suggest that the cloned gene of chicken Ii could be expressed in prokaryotic cells.
Key words:  chicken Ii gene,cloning,expression in prokaryotic cells

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