摘要
目的:克隆并原核表达小鼠高迁移率族蛋白1(high mobility group box1,HMGB1)。方法:从小鼠脾脏细胞中提取总RNA,应用RT-PCR获得小鼠HMGB1cDNA,经PCR扩增小鼠HMGB1基因;通过T-A克隆将该基因构建到PMD-18T载体中,测序鉴定;将该基因插入pET-28a(+)表达载体,IPTG诱导后,可表达相对分子质量约30 kDa的蛋白。蛋白质印迹鉴定表达的目的蛋白。结果:经RT-PCR扩增得到648 bp的DNA片段,序列分析显示与基因库中报道的已知序列完全一致;构建了含有HMGB1编码序列的原核表达载体,经诱导表达、蛋白质印迹鉴定,获得相对分子质量约30 kDa的融合蛋白。结论:成功克隆和表达了小鼠HMGB1基因,为HMGB1蛋白的功能试验奠定了基础。
Objective： To clone and express mouse high mobility group box chromosomal proteinl （HMGB1） gene. Methods： Total RNA was extracted from mouse spleen cells, and the whole length of HMGB1 gene was obtained by RT-PCR. The HMGB1 gene was cloned into PMD-18T vector by T-A clone and sequenced. Then the gene was inserted into pET-28a （ ＋ ） expression vector, identified by Western Blot. Results： The target DNA sequence of HMGB1 was obtained by RT-PCR which was about 648 bp length,sequencing results showed that HMGB1 gene was exactly consistent with the sequence reported in GenBank;Western Blot identified there was a protein strap about Mr. 30 000. The HMGB1 protein was successfully expressed. Conclusion： Clone and express mouse HMGB1 gene. It might provide a foundation for further study on the protein of HMGB1 function.
出处
《江苏大学学报（医学版）》
CAS
2009年第3期230-232,共3页
Journal of Jiangsu University：Medicine Edition
基金
国家自然科学基金资助项目(30871193
30710103093)
江苏省高等学校大学生实践创新训练计划
江苏大学大学生科研立项(06A080)
关键词
高迁移率族蛋白
克隆
原核表达
high mobility group boxl
clone
prokaryotic express
