摘要
目的表达纯化可溶性肿瘤坏死因子(tumor necrosis factor,TNF)相关凋亡诱导配体(TNF-related apoptosisinducing ligand,TRAIL)蛋白,并测定其生物学活性。方法利用E.coli中表达C-端带有6×His标签的可溶性TRAIL蛋白,并优化诱导时间(4、6和8 h)和诱导剂IPTG的浓度(0.06、0.125、0.25、0.5、1 mmol/L)。表达产物经Ni亲和层析和离子交换层析纯化后,采用MTT法测定不同浓度TRAIL(终浓度分别为100、200、400 ng/ml)的活性,并检测亲和层析过程中两种洗脱方式(洗脱液4℃留柱过夜后进行洗脱和洗脱液进柱后直接收集流出液)及纯化过程中两次透析的时间(均为24和48 h)对TRAIL活性的影响。结果最佳诱导时间为6 h,最佳IPTG诱导浓度为0.5 mmol/L。表达产物的表达量占菌体总蛋白的20%,相对分子质量为20 000,可与兔抗人TRAIL单克隆抗体发生特异性结合。TRAIL终浓度为100、200和400 ng/ml的实验组的抑制率分别约为36%、53%和73%。亲和层析的两种洗脱方式的TRAIL活性差异无统计学意义(P>0.05),透析48 h比透析24 h获得的TRAIL活性显著降低(P<0.05)。结论 TRAIL蛋白成功在E.coli中表达,具有抑制肿瘤细胞生长的作用,优化后的TRAIL诱导条件和纯化方法可进一步大量生产高TRAIL蛋白的活性,满足了TRAIL的基础及临床相关研究的要求。
Objective To express and purify soluble tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) and determine its biological activity. Methods Soluble TRAIL protein with 6 × His tag at C-terminus was expressed in E. coli, and the time for induction(4, 6 and 8 h) and IPTG concentration(0. 06, 0. 125, 0. 25, 0. 5 and1 mmol / L) were optimized. The expressed product was purified by Ni affinity and ion-exchange chromatography, of which the activities at various final concentrations(100, 200 and 400 ng / ml)were determined by MTT assay. The effects of two elution procedures(the eluant was maintained at 4 ℃ overnight in column before elution; the eluate was collected directly after the eluant was loaded to the column)and the time for dialysis(24 and 48 h) during purification on TRAIL activity were evaluated. Results The optimal time for induction was 6 h, while the optimal IPTG concentration was0. 5 mmol / L. The expression level of TRAIL protein was about 20% of total somatic protein, while the relative molecular mass was 20 000. The expressed product showed specific binding to rabbit anti-human TRAIL monoclonal antibody. The inhibiting rates of SW480 cells treated with TRAIL at final concentrations of 100, 200 and 400 ng / ml were about 36%,53% and 73% respectively. The elution procedure showed no significant effect on TRAIL activity(P 〉 0. 05). However,the TRAIL activity after dialysis for 48 h was significantly lower than that for 24 h(P 〈 0. 05). Conclusion Soluble TRAIL protein was successfully expressed in E. coli, which showed inhibiting effect on the growth of tumor cells. A large quantity of TRAIL may be produced under the optimized condition for induction by the optimized method for purification,which may meet the requirements for basic and clinical studies on TRAIL.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第10期1028-1032,共5页
Chinese Journal of Biologicals
基金
山西省自然科学基金(2012011041-5)
国家自然科学基金青年基金(81101895)
山西省高等学校优秀青年学术带头人支持计划[晋教科(2012)10号]
国家科技部重大新药创制计划(2012ZX09103301-010)