黄鳍棘鲷重组IL-1β的表达及生物学活性检测

Expression and bioactivity analysis of Acanthopagrus latus recombinant IL-1β

将扩增得到的黄鳍棘鲷Acanthopagrus latus白细胞介素1β(Interleukin 1β,IL-1β)基因克隆到表达载体pQE30,并转化到大肠杆菌M15中进行表达。采用Ni2+-Chelating Sepharose FF层析对重组蛋白进行纯化,并用梯度透析对纯化蛋白进行复性。经SDS-PAGE和Western Blot分析,获得了分子量为21kD的重组白细胞介素1β(Recombinant Interleukin 1β,rIL-1β)。采用Percoll不连续密度梯度离心分离黄鳍棘鲷头肾白细胞,在分离液密度1.080g/ml的界面上可以有效富集白细胞。用不同终浓度的rIL-1β和5μg/ml脂多糖(LPS)刺激培养的黄鳍棘鲷头肾白细胞,23℃培养4h后提取细胞的总RNA,经RT-PCR半定量检测,当培养基中rIL-1β的浓度达到20ng/ml时可以提高IL-1β基因的转录水平,与5μg/mlLPS的刺激效果相当,表达的重组蛋白表现出很好的生物学活性。

Interleukin-1β（IL-1β） gene was amplified from the total RNA of Acanthopagrus latus. The PCR product was subcloned into expression vector pQE30 and was subsequently transformed into E. coli M15 with IPTG inducement to be expressed. The recombinant protein was purified with Ni^2＋-chelating sepharose FF chromatography and the renaturation of the recombinant protein was subjected to gradient dialysis. The results of SDS-PAGE and Western Blot analysis proved that the recombinant IL-1β with a molecular weight of 21kD was successfully expressed. The head kidney leucocytes of A. latus were isolated by using a discontinuous Percoll density gradient centrifugation and the density of 1. 080g/ml was suitable for the isolation of head kidney leucocytes. The cultured head kidney leucocytes of the A. latus were stimulated by different final concentrations of rIL-1β and 5μg/ml LPS. The total RNA of the leucocytes was extracted after 4 h at 23℃. The RT-PCR revealed that rIL-1β could up-regulate the transcription of IL-1β at 20ng/ml and was equivalent to the effect of 5μg/ml LPS. The rIL-1β of A. latus showed efficacious bioactivity.