人IL-3基因原核表达载体的构建及表达

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。

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

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.