人内皮-单核活化多肽-2的重组表达及其活性鉴定

Expression of recombined human endothelial monocyte-activating polypeptideⅡand determination of its activity

目的:采用原核表达系统重组表达抗肿瘤细胞因子——人内皮-单核活化多肽2(endothelail monocyte-activating polypeptideⅡ,EMAP-Ⅱ),并探讨重组EMAP-Ⅱ的抗肿瘤生物学活性。方法:采用pMAL-p2x表达系统和BL-21菌株克隆表达人EMAP-Ⅱ_(147-312),对表达产物进行纯化。以发光底物法测定重组EMAP-Ⅱ介导人脐静脉内皮细胞ECV-304产生组织因子的活性;以流式细胞术测定其增强ECV-304细胞对于TNFα致凋亡的敏感性;以MTF法检测定其对胰腺癌SW1990细胞增殖的抑制效果。结果:测序证实EMAP-Ⅱ编码序列被正确克隆进表达载体,经诱导表达、纯化,获得重组EMAP-Ⅱ产物,每克菌体(湿重)可获得约500μg的重组蛋白,纯度为电泳纯。重组EMAP-Ⅱ在体外培养条件下可使ECV-304细胞产生TF因子;并可增强ECV-304细胞对于TNFα介导凋亡的敏感性,使凋亡率明显增加[(16.6±2.5)% vs(25.6±2.3)%,P<0.01];在一定的质量浓度(1μg/ml)下,EMAP-Ⅱ对SW1990细胞的增殖有一定抑制作用(P<0.05)。结论:本研究采用原核表达系统pMAL-p2x可表达高纯度的重组EMAP-Ⅱ,该因子具有良好的抗肿瘤生物学活性。

Objective： To clone and express the recombinant human endothelial monocyte-activating polypeptide-Ⅱ （EMAP-Ⅱ ） and identify its anti-tumor biological activities. Methods： EMAP-Ⅱ,47-3,2 was expressed by the expression vector pMAL -p2x and E. coli BL-21 and the product was purified. The production of tissue factor （TF） in human umbilical vein endothelial cell ECV-304 mediated by the recombinant EMAP-Ⅱ was determined by chemiluminescence substrate. The promoting effect of recombinant EMAP- Ⅱ on TNFα-induced ECV-304 cell. Apoptosis was determined by flow cytometry. Its inhibitory effect on human pancreaic cancer cell SW1990 proliferation was determined by MTT method. Results：DNA sequencing verified that EMAP-Ⅱ was correctly cloned. The molecular mass of the protein identified by SDS-PAGE was consistent with the theoretic value. The productivity of recombinant EMAP- Ⅱ was 500 μg per 1 g bacteria （wet mass）. The purified product induced expression of tissue factor（TF） in ECV-304 cells; it also enhanced the sensitivity of ECV-304 cells to the apoptotic effect of TNFα （ [ 16.6 ± 2.5 ] % vs [ 25.6 ±2.3 ] %, P 〈 0.01 ）, with the apoptotic rate reaching （ 25. 6 ± 2. 3 ） %. EMAP- Ⅱ protein （ 1 μg/ml ） inhibited the proliferation of SW1990 cells. Conclusion： The recombinant EMAP-Ⅱ can be cloned and expressed correctly in the pMAL-p2x expression system, and the recombinant product has effective anti-tumor effect.