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目的克隆表达CRIF1基因蛋白,为进一步研究该基因在骨髓微环境诱导白血病细胞G0/G1期阻滞过程中作用以及相关机制奠定基础。方法自HL60细胞中提取总RNA、逆转录,并与pET32a构建融合蛋白表达载体、蛋白原核表达载体;优化蛋白表达诱导条件,蛋白纯化;SDS-PAGE、Western Blot检测目的蛋白的表达。结果克隆并经测序获得全长744bp的CRIF1基因,成功构建融合蛋白表达载体和原核表达载体,优化并纯化蛋白;SDS-PAGE在42.5 kD处获得目的条带,Western Blot检测确定为CRIF1表达蛋白。结论成功克隆表达出CRIF1基因蛋白。
Objective To clone and express CRIF1 gene protein and lay a foundation for further study of the role of this gene in G0 / G1 phase arrest induced by bone marrow microenvironment in leukemic cells. Methods The total RNA was extracted from HL60 cells and reverse transcribed. The fusion protein expression vector and prokaryotic expression vector were constructed with pET32a. The conditions of protein expression and protein purification were optimized. The expression of target protein was detected by SDS-PAGE and Western Blot. Results The full-length 744-bp CRIF1 gene was cloned and sequenced. The fusion protein expression vector and prokaryotic expression vector were successfully constructed, and the protein was optimized and purified. The target band was obtained at 42.5 kD by SDS-PAGE and CRIF1 protein was identified by Western Blot. Conclusion The CRIF1 gene protein was successfully cloned and expressed.