摘要
用Trizol提取人骨髓总RNA,通过RT PCR扩增人干细胞因子DNA,克隆到pMD18 T载体中并测序将其定向连入原核表达载体pET32a(+),获得了重组表达载体pET32a(+)/hSCF,其cDNA长度为504bp,测序结果与已知序列吻合然后在大肠杆菌BL21中经IPTG诱导表达,获得37kD的融合蛋白,约占菌体总蛋白量的30% 经Ni2+
Total RNA was extracted from fresh marrow by Trizol Reagent, and human stem cell factor cDNA was amplified by RT- PCR , then cloned into vector pMD18 T for sequencing. Prokaryotic expression vector pET32a (+ ) / SCF was constructed and expressed in E. coli BL21 with induction of IPTG. The fusion protein was purified with Ni^(2+)-NTA agarose resin. In this study, recombinant stem cell factor cDNA cloned was 500bp in length. DNA sequencing showed the DNA sequence of the cloned gene was the same as the published sequence. SDS-PAGE analysis showed the prokaryotic expression vector pET32a( + ) / hSCF fusion protein with relative molecule mass 37kD was expressed in E. coli BL21,and the amount of it is about 30% of the total bacterial protein after being induced by IPTG for 3h. The product was obtained with a purity of about 90%.
出处
《四川大学学报（自然科学版）》
CAS
CSCD
北大核心
2005年第3期584-587,共4页
Journal of Sichuan University(Natural Science Edition)
