大肠杆菌Nissle1917 L-天冬酰胺酶Ⅱ基因在大肠杆菌BL21中的表达与抗肿瘤活性

Expression of Escherichia coli Nissle1917 L-asparaginaseⅡ-encoding gene in E. coli BL21 and anti-tumor activity

摘要:

【目的】从大肠杆菌Nissle1917中获得L-天冬酰胺酶Ⅱ基因，并研究其抗肿瘤活性。【方法】以大肠杆菌Nissle1917基因组为模板PCR扩增L-天冬酰胺酶Ⅱ基因，克隆至可诱导表达载体pET28a上。将L-天冬酰胺酶Ⅱ表达载体pET28a-asp转化至大肠杆菌BL21(DE3)中并通过IPTG诱导表达，经聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相色谱-质谱(LC-MS)对表达的L-天冬酰胺酶Ⅱ进行鉴定，并通过镍柱亲和层析纯化收集表达出的L-天冬酰胺酶Ⅱ。用纯化定量以后的L-天冬酰胺酶Ⅱ作用小鼠乳腺癌4T1细胞、人肝癌Hep-3B细胞和人脐静脉内皮细胞HUVEC。【结果】来自于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ基因可在大肠杆菌BL21中高效表达并通过LC-MS得到鉴定，细胞毒性实验结果表明L-天冬酰胺酶Ⅱ对4T1细胞和Hep-3B细胞的生长具有较强的抑制作用，而对人脐静脉内皮细胞HUVEC的生长无明显抑制效果。【结论】来源于大肠杆菌Nissle1917的L-天冬酰胺酶Ⅱ能显著抑制4T1细胞和Hep-3肿瘤细胞的生长，而对人正常组织细胞的生长无明显抑制效果，为进一步研究L-天冬酰胺酶Ⅱ特异性抗肿瘤作用机制和对实体瘤的应用研究奠定了重要基础。

Abstract:

[Objective] To study the anti-tumor activity of L-asparaginase II (ASP) of Escherichia coli Nissle1917. [Methods] L-AsparaginaseⅡ gene was amplified from the genome of E. coli Nissle1917 and inserted on the expression vector pET28a. The final expression vector pET28a-asp was transformed into E. coli BL21(DE3) for ASP expression. SDS-PAGE and LC-MS were used to determine the correct expression of ASP. After purification by nickel-affinity chromatography, the recombinant ASP was used to treat mouse 4T1 breast tumor cells, Hep-3B human hepatoma cells and Human Umbilical Vein Endothelial Cells (HUVEC). [Results] L-AsparaginaseⅡgene from E. coli Nissle1917 was successfully expressed in E. coli BL21(DE3) and identified by LC-MS. The purified L-asparaginaseⅡinhibited the growth of 4T1 and Hep-3B tumor cells while not HUVEC. [Conclusion] The L-asparaginaseⅡ of E. coli Nissle1917 was active on 4T1 and Hep-3B tumor cells while not on normal tissue cells. Our results will be helpful for the further study on the active mechanism of ASP and its application in solid tumors therapy.