摘要
目的构建肿瘤转移相关基因1短式(MTA1s)原核表达载体并诱导其蛋白表达,为后续MTA1s的相关研究奠定基础。方法取对数生长期的乳腺癌MCF-7细胞,提取细胞总RNA并反转录成cDNA,并以此为模板扩增MTA1s基因的蛋白编码区。采用一步克隆的方法将目的片段插入表达载体pET-28a(+)中并构建重组原核表达载体(命名为:pET-28a-MTA1s),将构建的重组载体用双酶切、菌液聚合酶链式反应(菌液PCR)、和测序进行鉴定。将pET-28a-MTA1s导入细菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达MTA1s重组蛋白,通过SDS-PAGE进行鉴定重组蛋白(MTA1s)表达情况以及蛋白免疫印迹分析(Western blot)鉴定重组蛋白(MTA1s)表达量。结果以MCF-7乳腺癌细胞为模板扩增出大小位置正确的片段;构建的pET-28a-MTA1s重组载体经双酶切、菌液PCR验证正确,DNA测序证实序列正确。考马斯亮蓝染胶和Western blot结果显示IPTG诱导表达的重组蛋白分子量正确且条带单一。结论成功构建pET-28a-MTA1s原核表达载体,为后续进行下游实验的研究奠定基础。
Objective To construct the prokaryotic expression vector of tumor metastasis related gene 1 short form MTA1s and induce its protein expression,so as to lay a foundation for the follow-up study of MTA1s.Methods The total RNA of breast cancer MCF-7 cells in logarithmic phase was extracted and inversely transcribed into cDNA,which was used as a template to amplify the protein coding region of MTA1s gene.The target fragment was inserted into the expression vector pET-28a(+)by one-step cloning and the recombinant prokaryotic expression vector named pET-28a-MTA1s was constructed.The constructed recombinant vector was identified by double enzyme digestion,bacterial liquid polymerase chain reaction(PCR)and sequencing.pET-28a-MTA1s was introduced into bacterial BL21(DE3).The expression of MTA1s recombinant protein was induced by isopropyl-β-D-thiogalactoside(IPTG).The expression of recombinant protein(MTA1s)was identified by SDS-PAGE and the expression of recombinant protein(MTA1s)was identified by Western blot.Results The fragments with correct size and position were amplified from MCF-7 breast cancer cells,and the constructed pET-28a-MTA1s recombinant vector was verified by double enzyme digestion and bacterial liquid PCR,and the sequence was confirmed by DNA sequencing.The results of Coomassie brilliant blue staining and Western blot showed that the molecular weight of the recombinant protein induced by IPTG was correct and the band was single.Conclusion The prokaryotic expression vector of pET-28a-MTA1s is successfully constructed,which lays a foundation for the further study of downstream experiments.
作者
刘洋
王攀琦
王芳
刘星吟
董兰
魏瑶璐
宁志丰
沈定文
LIU Yang;NING Zhi-feng;SHENG Ding-wen(School of Pharmacy,Xianning Medical College,Hubei University of Science and Technology,Xianning Hubei 437100,China)
出处
《湖北科技学院学报（医学版）》
2022年第2期118-124,共7页
Journal of Hubei University of Science and Technology(Medical Sciences)
基金
湖北科技学院国培项目(2019-21GP04)
五官专项(2019-1,2020-2)。
关键词
肿瘤转移相关基因
原核表达
同源重组
载体构建
Tumor metastasis related genes
Prokaryotic expression
Homologous recombination
Vector construction
