人IL-3基因原核表达载体的构建及表达

Construction and Expression of the hIL-3-Prokaryotic Expression Vector

目的:构建人IL-3基因原核表达载体pET32a-IL-3,并在大肠杆菌中诱导表达。方法:通过佛波酯(TPA)和植物球血凝素(PHA)刺激人T淋巴细胞系Jurkat细胞,增加IL-3mRNA表达水平,提取mRNA,逆转录-聚合酶链反应(RT-PCR)获得cDNA,以Jurkat细胞cDNA为模板,通过PCR方法扩增得到人IL-3基因,将其克隆入原核表达载体pET32a(+)中,将重组质粒转化入大肠杆菌宿主菌株BL21中,以异丙基硫代半乳糖苷(IPTG)诱导融合蛋白表达,并通过改变IPTG浓度,诱导时间,诱导温度等条件最终实现蛋白的可溶性表达。表达产物用SDS-PAGE检测表达情况。结果:酶切鉴定和测序结果证明成功构建了原核表达载体pET32a-IL-3。SDS-PAGE检测结果证明实现了人IL-3基因在大肠杆菌中的可溶性表达。结论:成功构建了人IL-3基因的原核表达载体并在大肠杆菌中获得了良好的表达。

Objective： To construct the prokaryotic expression vector of human IL-3 pET32a-IL-3, and to expresse the prutein in Escherichia coli. Methods： The human T cell leukemia cell line Jurkat cells were stimulated by phorbol ester（TPA） and phytohaemagglutinin （PHA） to increase the expression of IL-3mRNA. The cDNA was got by using the RT-PCR. The human IL -3 gene was amplified by polymerase chain reaction （PCR） with the cDNA of Jurkat cells as the template. The amplified fragment was cloned into the pET32a（＋） vector, and transformed into the E. coli BL21. Isopropyl-D-thiogalactopyranoside （IPTG） was used to induce the protein expression. By changing the IPTG concentration, induction time, induction temperature, the soluble expression of the protein were ultimately implemented. SDS-PAGE was used to analyze the expression products. Results： The prokaryotic expression vector pET32a-IL-3 were successfully constructed. The soluble expression of human IL-3 in Escherichia coli was implemented. Conclusions： The prokaryotic expression vector pET32a-IL-3 was successfully constructed.