重组鼠干细胞因子的原核表达、纯化及生物活性初步分析

Prokaryotic Expression, Purification and Primary Bioactivity Assay for Recombinant Mouse Stem Cell Factor

目的:研制重组鼠干细胞因子(rmSCF),并检测其体外生物学活性。方法:提取BALB/c孕龄鼠总RNA,通过RT-PCR扩增mSCF的编码基因,将其克隆至原核表达载体pBV220,转化大肠杆菌DH5α,将重组菌株接种至氨苄青霉素抗性的LB培养基中,扩大培养至5L发酵罐中,42℃诱导表达,经包涵体复性、DEAE-SepharoseFF和Phenyle-SepharoseHP层析纯化,制备rmSCF。结果:构建了pBV220-mSCF原核表达载体,在大肠杆菌中表达了rmSCF,纯化后的rmSCF对类原巨核细胞白血病细胞系的UT-7细胞有明显的刺激作用,比活为1.0×106U/mg。结论:获得了纯度大于95%的rmSCF,将进一步展开其在小鼠体内的药效学研究。

Objective： To express and purify recombinant mouse stem cell factor（nnSCF） in Escherichia coli, and analyze its bioactivity in vivo. Methods： Total mRNA was extracted from the embryonic liver from pregnant BALB/c mice, from which the gene encoding mSCF was amplified by RT-PCR. The PCR product was confirmed by sequence analysis, and was cloned into pBV220 to construct expression plasmid pBV220-mSCF, and then it was transformed into E.coli DH5oc The recombinant strain was inoculated into LB with ampicillin resistance and scaled up to the 5 liter fermentor, the recombinant mSCF protein was expressed when induced at temperature of 42℃. The rmSCF expressed as the inclusion body was renatured and purified by DEAE-sepharose FF and phenyle-sepharose HP chromatography. Its bioactivity of proliferation stimulation of megakaryocytic leukemic cell UT-7 was analyzed. Results： The prokaryotic expression vector pBV220- mSCF was constructed and rmSCF was expressed in E.coli efficiently. The purified rmSCF stimulated megakaryocytic leukemia cell UT-7 proliferation and its specific activity was 1.0×10^6 U/rag. Conclusion： The rmSCF with a purity of 95% was produced, and it will be subjected to the pharmacodynamic study in mice.