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目的:构建人TAT-Survivin融合蛋白原核表达载体。方法:以人胸腺细胞瘤cDNA为模板,采用RT-PCR扩增survivin基因成熟蛋白编码的全部序列,克隆入原核表达载体pET28a(+)中,经限制性内切酶双酶切及DNA序列分析鉴定目的基因。结果:PCR扩增的特异性片段长度为462bp,以此构建的重组质粒pET28a-TAT-survivin,经HindIII和BamHI双酶切后显示5.9kb和462bp左右的两条片段,测序结果与Genbank中的人survivin基因cDNA(Genbank序列号)序列一致。证明人survivin已成功克隆到了原核细胞表达载体pET28a(+)中。结论:成功构建了pET28a-TAT-survivin重组原核表达载体。
Objective: To construct prokaryotic expression vector of human TAT-Survivin fusion protein. Methods: Human thymoma cell cDNA was used as a template to amplify the full-length sequence of survivin gene by RT-PCR and cloned into the prokaryotic expression vector pET28a (+). The recombinant plasmid was digested by restriction enzyme and analyzed by DNA sequence Identification of the purpose of the gene. Results: The specific fragment length of PCR amplification was 462bp. The recombinant plasmid pET28a-TAT-survivin was digested by HindIII and BamHI and showed two fragments of 5.9kb and 462bp. The sequencing results were in good agreement with those of GenBank Human survivin gene cDNA (Genbank sequence number) sequence. Proved that human survivin has been successfully cloned into the prokaryotic expression vector pET28a (+). Conclusion: The recombinant prokaryotic expression vector pET28a-TAT-survivin was successfully constructed.