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目的构建VHL蛋白(von Hippel-Lindau protein,pVHL)β结构域肾癌抑制融合肽cDNA,并将其应用于肾癌的微基因治疗。方法分别设计合成pVHLβ结构域104-123短肽cDNA的正向和反向引物以及编码TAT-6×His cD-NA的正、反向引物,使用互为引物模板PCR方法,扩增获得具有NaeI/Eco72I酶识别位点的TAT-6×His cDNA片段和具有Eco72I/BamH I酶识别位点的VHLβ抑制肽cDNA片段,将两个片段分别克隆到pGEM-T easy中,使用双脱氧终止法测序,通过DNASIS软件分析同数据库资料一致性和编码的氨基酸序列。用NaeI和BamH I双酶切获取的TAT-6×His-VHLβcDNA片段插入到具有NT4信号肽的pBV220载体中,构建了pBV220-NT4-TAT-6×His-VHLβ质粒。结果经DNA测序证实,获得了编码TAT-6×His cDNA片段和编码VHLβ抑制肽的cDNA片段,分析证实核酸序列和推导的氨基酸同数据库资料一致。重组质粒pBV220-NT4-TAT-6×His-VHLβ酶切图谱证实已将NT4-TAT-6×His-VHLβ融合肽cDNA克隆到pBV220原核表达载体中。结论成功扩增了编码TAT膜渗透肽和6×His标签肽以及VHL抑癌基因β结构域肾癌抑制肽的cDNA片段,构建了具有NT4信号肽的TAT-6×His-VHLβ融合肽cDNA。
OBJECTIVE: To construct von Hippel-Lindau protein (pVHL) β-domain renal cell carcinoma (RGC) cDNA fusion protein and to apply it to the micro-gene therapy of renal cell carcinoma. Methods The forward and reverse primers of the pVHLβ domain 104-123 short peptide cDNA and the forward and reverse primers of TAT-6 × His cD-NA were designed and synthesized respectively. The PCR products with NaeI / Eco72I enzyme recognition site of the TAT-6 × His cDNA fragment and a Eco72I / BamH I enzyme recognition site VHLβ inhibitor peptide cDNA fragment, the two fragments were cloned into pGEM-T easy sequencing using dideoxy termination DNA sequences were analyzed by DNASIS software for identity and coding of the same database. The pBV220-NT4-TAT-6 × His-VHLβ plasmid was constructed by inserting the TAT-6 × His-VHLβ cDNA fragment digested with NaeI and BamH I into the pBV220 vector with the NT4 signal peptide. Results DNA sequencing confirmed that cDNA encoding TAT-6 × His cDNA fragment and VHLβ inhibitory peptide was obtained. The analysis confirmed that the nucleotide sequence and the deduced amino acid were consistent with the database. The restriction map of recombinant plasmid pBV220-NT4-TAT-6 × His-VHLβ confirmed that the cDNA of NT4-TAT-6 × His-VHLβ fusion peptide was cloned into pBV220 prokaryotic expression vector. Conclusion The cDNA fragment of TAT-6 × His-VHLβ fusion peptide with NT4 signal peptide was constructed by amplifying the cDNA fragment coding for TAT membrane permeable peptide and 6 × His tag peptide and the β-domain renal cell carcinoma inhibitory peptide of VHL tumor suppressor gene.