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.