兰州大学机构库 >学院待认领
人PKM2基因克隆、表达及蛋白纯化
Alternative TitleCloning, expression and protein purification of M2-type pyruvate kinse
王秋香
Thesis Advisor王海琳
2013-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
KeywordPKM2 克隆 表达 纯化
Abstract目的 构建人丙酮酸激酶基因PKM2 (M2-type pyruvate kinse)的原核表达质粒pET30a(+)-PKM2,在大肠杆菌DH5α中进行基因扩增,导入大肠杆菌BL21中进行人丙酮酸激酶基因PKM2融合蛋白表达,镍离子柱亲和层析法纯化融合蛋白。 方法 用人Hela细胞提取RNA,反转录获得人cDNA。以cDNA为模板,用PCR方法扩增出目的基因PKM2,用限制性内切酶(BamHⅠ﹑SalⅠ及NdeⅠ﹑SalⅠ)对目的片段及质粒pET30a(+)进行双酶切,及T4DNA连接酶进行连接,并将它们依次插入到表达载体pET30a(+)的多克隆位点中构建重组质粒pET30a(+)- PKM2。经限制性内切酶(BamHⅠ﹑SalⅠ及NdeⅠ﹑SalⅠ) 进行双酶切和DNA测序证实正确后,将此质粒转化入大肠杆菌BL-21(DE3)中表达融合蛋白PKM2,用Ni-NTA亲和层析柱进行纯化,聚丙烯酰胺凝胶电泳(SDS-PAGE),考马斯亮兰染色,在凝胶成像分析系统中分析纯化后蛋白情况。 结果 通过PCR方法,成功获得目的基因片段。经BamHⅠ﹑SalⅠ及NdeⅠ﹑SalⅠ双酶切鉴定所切下的片段大小与理论值相符,北京六合华大基因科技服务有限公司测序结果和报道一致。经SDS-PAGE分析,导入质粒载体pET30a(+)- PKM2后,大肠杆菌BL21成功大量表达PKM2融合蛋白;Ni-NTA亲和层析柱纯化后的蛋白约在58 KDa位,条带单一,无杂带出现。 结论 成功构建了人PKM2基因的扩增质粒载体pET30a(+)- PKM2;成功表达纯化了PKM2融合蛋白,为肿瘤疫苗研制和肿瘤标记物快速诊断的研究奠定基础。
Other AbstractObjective:To constract the pET30a(+)-PKM2 expression plasmid , amplifiy this plasmid in E.clio DH5α,express and purify the fusion proteion in E.clio BL21,the recombinant 6×His fused protein was perified by Ni-NTA purification. Methods:Extract RNA from Hela cell,and then reverse transcriptase the RNA,we can get cDNA.The PKM2 gene was amplified by PCR from cDNA template.Use restriction enzyme(BamHⅠ﹑SalⅠand NdeⅠ﹑SalⅠ)digestion PKM2 and pET30a(+),get recombination plasmid pET30a(+)- PKM2 by T4 DNA ligase. Restriction enzyme digestion and sequence recombination plasmid pET30a(+)- PKM2 , PKM2 sequence is correct.Express and purify the fusion proteion in E.clio BL21,the recombinant 6×His fused protein was perified by Ni-NTA purification and dialysis method.The recmbination proteion electrophoresis get through SDS-PAGE,decoration by Coomassie brilliant blue method ,analyzed by gel imaging and analysis system. Results:Successed get PKM2 through PCR.The recombination plasmid pET30a(+)- PKM2 were identified by restriction enzyme digestion and nucleotide sequencing.The result of the recombination proteion electrophoresis get through SDS-PAGE,show E.clio BL21 can express recombination proteion PKM2. After Ni-NTA and dialysis method purification,the molecular weight of the recombination proteion PKM2 approximately 58 KDa. preparations of the recombination proteion PKM2 showed a single diffuse band on SDS-PAGE. Conclusion: The successfully constract the pET30a(+)- PKM2 expression plasmid. Recmbination proteion PKM2 is succeed expressed and purified,which can provide basis for research of tumor vaccine and tumor diagnosis.
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Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/222810
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
王秋香. 人PKM2基因克隆、表达及蛋白纯化[D]. 兰州. 兰州大学,2013.
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