文章摘要
楼秋雯,陈为为,黄志广,等.丝瓜籽核糖体失活蛋白Luffin-α的基因克隆、表达及抗肿瘤活性研究[J].浙江中医药大学学报,2020,44(8):707-714.
丝瓜籽核糖体失活蛋白Luffin-α的基因克隆、表达及抗肿瘤活性研究
Cloning, Expression and Antitumor Activity of Luffin-α from the Seeds of Luffa Cylindrica
投稿时间:2020-02-03  
DOI:10.16466/j.issn1005-5509.2020.08.001
中文关键词: Luffin-α基因  丝瓜  克隆  IPTG浓度  诱导温度  可溶性表达  重组蛋白  抗肿瘤活性
英文关键词: Luffin-α gene  Luffa cylindrica  cloning  IPTG concentrations  induction temperature  soluble expression  recombinant protein  anti-tumor activity
基金项目:
作者单位E-mail
楼秋雯 浙江中医药大学生命科学学院 杭州 310053  
陈为为 浙江中医药大学生命科学学院 杭州 310053  
黄志广 浙江中医药大学生命科学学院 杭州 310053  
安紫珲 浙江中医药大学生命科学学院 杭州 310053  
朱振洪 浙江中医药大学生命科学学院 杭州 310053 zhenhongzhu@aliyun.com 
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中文摘要:
      [目的]克隆丝瓜籽Luffin-α基因,优化其在大肠杆菌中可溶性表达的条件,并进行初步的抗肿瘤活性测定。[方法]采用反转录PCR(reverse transcription polymerase chain reaction,RT-PCR)的方法克隆获得丝瓜籽Luffin-α基因,构建pGEX6P-1/Luffin-α表达载体,并转化至大肠杆菌BL21(DE3)中获得重组菌株,摸索不同异丙基-β-D-硫代半乳糖苷(isopropyl-β-D-thiogalactopyranoside,IPTG)浓度、诱导温度对重组蛋白可溶性表达的影响。以谷胱甘肽硫转移酶(glutathione-S-transferase,GST)亲和层析方法纯化目标蛋白,并使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)和Western blot对其进行检测及鉴定。采用四甲基偶氮唑盐微量酶反应比色法(methylazo salt trace enzyme reaction colorimetric assey,MTT)探讨其抗肿瘤活性。[结果]成功克隆丝瓜籽Luffin-α基因,全长834bp。优化后的可溶性蛋白表达条件为16℃,0.8mmol·L-1的IPTG诱导20h。SDS-PAGE和Western blot分析鉴定可见相对分子质量约为56kDa的特异性条带。经MTT法检测,重组蛋白对肝癌细胞HepG2和乳腺癌细胞MDA-MB-231均有显著的抑制效果。[结论]成功建立Luffin-α大肠杆菌表达系统,并优化表达条件,初步鉴定重组蛋白具有较好的抗肿瘤活性,为后续相关药物的开发奠定基础。
英文摘要:
      [Objective] To clone the Luffin-α gene from the seeds of Luffa cylindrical, optimize its soluble expression in E.coli, and conduct preliminarily anti-tumor activity assay.[Methods] The Luffin-α gene was cloned by reverse transcription polymerase chain reaction(RT-PCR) to construct pGEX6P-1/Luffin-α expression vector, and then converted to E. coliBL21(DE3) to obtain recombinant strains. Isopropyl-β-D-thiogalactopyranoside(IPTG) concentrations and induction temperature were explored to acquire the optimizational soluble expression conditions of recombinant protein. The target protein was purified by glutathione-S-transferase(GST) affinity chromatography and it was detected and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot. Finally, the antitumor activity was evaluated by methylazo salt trace enzyme reaction colorimetric assay(MTT) method.[Results] The Luffin-α gene with a total length of 834bp was cloned successfully. The optimized expression conditions were achieved by induction with 0.8mmol·L-1 IPTG for 20h at 16℃. SDS-PAGE and Western blot analysis identified specific bands with a relative molecular weight of about 56kDa. The purified protein significantly inhibited proliferation of hepatoma carcinoma cell HepG2 and mammary cancer cell MDA-MB-231 by MTT assay.[Conclusion] The E.coli expression system of Luffin-α and optimizational expression conditions have been successfully established, and strong anti-tumor activity of recombinant protein has been preliminarily identified, which lays a foundation for the subsequent development of related drugs.
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