| 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 |
| 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 |
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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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