鼠高迁移率族蛋白1A盒基因的克隆和原核表达

目的:克隆小鼠高迁移率族蛋白1A盒(HMGB1 A box)cDNA,并在大肠杆菌中表达GST-A盒融合蛋白。方法:从鼠肺提取总RNA,经RT-PCR获得HMGB1 A盒基因。将该基因插入pGEX-4T-2载体的BamHI和EcoRI位点之间并测序鉴定,转化BL21(DE3)后30℃IPTG诱导表达5h,进行SDS-PAGE分析。结果:DNA测序证明,获得了HMGB1 A盒基因,其序列与GenBank中报道序列基本一致。SDS-PAGE分析表明,HMGB1 A盒与GST融合蛋白获得成功表达,分子质量约36KD,表达量约占菌体总蛋白的15%,结论:通过RT-PCR成功克隆和表达了鼠HMGB1 A盒基因。

Cloning and expression of mouse HMGB1 a box gene

Objective: To clone, express mouse High mobility group box chromosomal protein 1(HMGB1) A box gene. Methods: Total RNA was extracted from the lung tissue of a new born mouse, and the HMGB1 A box gene was obtained by RT-PCR using specific primers. Then the A box gene was cloned into BamHI and EcoRI site of pGEX-4T-2 expression vector and confirmed by sequencing. After transforming E.coli BL21(DE3), the recombinant bacteria was induced at 30℃ for 5h, the fusion protein GST-A box was analyzed by SDS-PAGE. Results: DNA sequencing result showed that HMGB1 A box gene was exactly consistent with the sequence reported in GenBank. SDS-PAGE analysis demonstrated that HMGB1 A-GST protein was expressed in E.coli BL21(DE3), and the molecular mass of it is 36 KD. The protein band amounted to 15% of total bacteria protein. Conclusion: Mouse HMGB1 A box gene was successfully cloned and expressed.