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目的:克隆人组蛋白H1全长编码区基因,获得其原核表达产物,并对融合蛋白进行纯化以及活性检测。方法:采用PCR技术从人乳腺文库中扩增出人组蛋白H1全长编码区基因,将其克隆到pGEX-KG载体中,在大肠杆菌Rossate中表达后,利用GST-Sepharose 4B亲和珠对原核表达产物进行纯化,以SDS-PAGE和Western blot鉴定表达与纯化产物。结果:从人乳腺文库中扩增获得约650 bp的DNA片段,并成功克隆至pGEX-KG载体上,经测序与目的序列完全一致;在Rossate菌中诱导表达出相对分子质量(Mr)约为52 000的目的蛋白,经纯化后获得了纯度较高的重组蛋白GST-H1,激酶实验证明该蛋白活性良好。结论:成功获得了活性良好的重组蛋白GST-H1,为后续研究细胞周期蛋白调控奠定了实验基础。
OBJECTIVE: To clone the full-length coding region of human histone H1 and obtain its prokaryotic expression product. The fusion protein was purified and its activity assayed. METHODS: Human Histone H1 full-length coding region gene was amplified from human breast cDNA library by PCR and cloned into pGEX-KG vector. After expressed in E. coli Rossate, GST-Sepharose 4B affinity beads The prokaryotic expression product was purified and the expression and purification products were identified by SDS-PAGE and Western blot. Results: A 650 bp DNA fragment was amplified from the human breast cDNA library and successfully cloned into pGEX-KG vector. The sequence was consistent with the target sequence. The relative molecular mass (Mr) 52 000 of the target protein purified to obtain a higher purity of the recombinant protein GST-H1, kinase experiments show that the protein activity is good. CONCLUSION: GST-H1, a well-established recombinant protein, has been successfully obtained and laid the foundation for subsequent studies on the regulation of cyclin.