| 人PKM2基因克隆、表达及蛋白纯化 |
| Alternative Title | Cloning, expression and protein purification of M2-type pyruvate kinse
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| 王秋香 |
| Thesis Advisor | 王海琳
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| 2013-05-30
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | PKM2
克隆
表达
纯化
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| 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 Abstract | Objective: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. |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/222810
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| Collection | 学院待认领
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| Affiliation | 临床医学院
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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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