胰岛素样生长因子1受体α亚单位基因重组真核表达质粒的构建

Construction of recombinant eukaryotic expression plasmid encoding IGF-1R α subunit gene

目的构建编码胰岛素样生长因子1受体(IGF-1R)α亚单位基因的重组真核表达质粒。方法根据GenBank中人IGF-1Rα亚单位基因编码序列设计并合成引物,用TRIzol一步法从Graves病患者甲状腺组织中提取总RNA,逆转录成cDNA并将其作为模板,PCR扩增获得IGF-1Rα亚单位编码基因片段。胶回收纯化后再与pcDNA^(TM)3.1D/V5-His-TOPO载体通过拓扑克隆连接,用热休克法将重组质粒转入大肠杆菌,在含有氨苄青霉素培养基中筛选阳性克隆。用碱裂解法提取IGF-1Rα-pcDNA^(TM)3.1D/V5-His-TOPO真核表达质粒,并对其进行PCR、限制性内切酶(HindⅢ、BamHⅠ、ScaⅠ)消化、DNA测序分析等鉴定。结果Graves病患者甲状腺组织中总RNAOD_(260)/OD_(280)为1.8~2.0,纯度较好且结构完整。经逆转录、PCR扩增出2679 bp的目的基因片段。挑取3个阳性克隆菌落提取质粒,PCR均扩增出目的片段;单酶切、双酶切后均得到相应长度的酶切产物,证明重组质粒中含有IGF-1Rα单位基因序列。经进一步测序、序列比对分析,重组质粒中的IGF-1Rα序列与NCBI公布序列完全一致,与载体接口连接无误,IGF-1Rα亚单位基因翻译读码框架正确。结论IGF-1Rα-pcDNA^(TM)3.1D/V5-His-TOPO重组真核表达质粒构建并鉴定成功。

Objective To construct a recombinant eukaryotic expression plasmid encoding insulin-like growth factor 1 receptor(IGF-1R) α subunit gene.Methods The primers were designed and synthesized according to the coding sequence of human IGF-1R α subunit gene in GeneBank.The total RNA was extracted from thyroid tissues in patients with Graves’ disease by one-step TRIzol method and reversely transcribed into cDNA,which was then taken as the template.After purification,the IGF-1R alpha subunit coding gene was amplified by PCR and ligated through topological cloning with pcDNA 3.1D/V5-His-TOPO vector to construct IGF-1R α-pcDNA^(TM) 3.1D/V5-His-TOPO eukaryotic expression plasmid.The recombinant plasmid was transformed into E.coli by heat shock method,and the positive clones were screened in the medium containing ampicillin.The eukaryotic expression plasmid of IGF-1R α-pcDNA^(TM)3.1D/V5-His-TOPO was extracted by alkaline lysis and identified by PCR,restriction enzyme digestion(Hind Ⅲ,BamH Ⅰ and Sca Ⅰ) and DNA sequence analysis.Results The total RNA in thyroid tissues of patients with Graves’ disease was pure and complete(OD260/OD280:1.8-2.0).A total of 2 679 bp fragment of target gene was amplified by reverse transcription and PCR.Three positive clone colonies were selected to extract plasmids,and the target fragments were amplified by PCR.The digested products of corresponding length were obtained after single enzyme digestion and double enzyme digestion,which proved that the recombinant plasmids contained IGF-1R α subunit gene sequence.Further sequencing and sequence alignment analysis showed that the IGF-1Rα subunit gene sequence in the recombinant plasmid was completely consistent with the published sequence of NCBI,and the connection with the vector interface was correct.The translation and reading frame of IGF-1R α subunit gene was correct.Conclusion The recombinant eukaryotic expression plasmid IGF-1R α-pcDNA^(TM)3.1D/V5-His-TOPO was successfully constructed and identified.