重组鼠干细胞因子的原核表达、纯化及生物活性初步分析

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。

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

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.