大肠杆菌Nissle1917L-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

作　　者：梁毅偈 孙运军[1] 胡胜标[1] 颜富 张旭[1] 白利明[1] 张友明[1] 丁学知[1] 夏立秋[1] 

机构地区：[1]湖南师范大学生命科学学院微生物分子生物学国家重点实验室培育基地,湖南长沙410081

出　　处：《微生物学通报》2014年第4期607-613,共7页Microbiology China

基　　金：国家973计划项目(No.2011CB227300);国家自然科学基金项目(No.31070006);高校博士学科点专项科研基金项目(No.20124306110006)

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。[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-Asparaginase Ⅱ gene from E. coli Nisslel 917 was successfully expressed in E. coli BL21 （DE3） and identified by LC-MS. The purified L-asparaginase Ⅱ inhibited the growth of 4T1 and Hep-3B tumor ceils while not HUVEC. [Conclusion] The L-asparaginase U of E. coli Nissle1917 was active on 4T1 and Hep-3B tumor ceils 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.

关 键 词：L-天冬酰胺酶Ⅱ 大肠杆菌 克隆 抗肿瘤 

分 类 号：Q78[生物学—分子生物学]