NT4 TAT 6×His VHLβ结构域肾癌抑制融合肽cDNA克隆的构建

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 cDNA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Nae I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。

Construction and identification of recombinant plasmid expressing NT4 TAT 6×His VHLβ fusion peptide

The specific amino acid sequence(104 123) in β domain of pVHL is sufficient to inhibit the growth and invasion of renal cell carcinoma（RCC）. To express this peptide for a gene therapy of RCC by using minigene, we constructed and identified the recombinant plasmid expression box, including NT4 signal peptide, tag and cell penetrating peptides. MethodsBy means of asymmetrical primer/template, fragments encoding β domain of pVHL(the VHL gene product) and TAT 6×His were gained, which included Nae I/Eco72I and Eco72I/BamH I restriction enzyme sites. Then the fragments were cloned into vector pGEM T easy. The positive clone was identified by restriction enzymes, and then the cloned amplified fragments were sequenced by a dideoxy mediated chain termination method. The cloned TAT 6×His VHLβ cDNA was compared with the GeneBank sequence by DNASIS. After being digested by Nae I and BamH I, the TAT 6×His VHLβ cDNA fragment was cloned into recombinant vector pBV220 NT4, which was also digested by Nae I and BamH I. The constructed recombinant plasmid pBV220 NT4 TAT 6×His VHLβ was obtained. ResultsThe sequences of TAT 6×His and VHLβ cDNA artificially synthesized and analyzed by digestion were consistent with the published results. After analyzing ORF of the cloned TAT 6×His VHLβ cDNA by DNASIS, we found that amino acids encoded by the cloned TAT 6×His VHLβ cDNA were also identical to the published results. ConclusionThe recombinant plasmid expressing TAT cell penetrating peptides and β domain of pVHL was constructed. Then prokaryotic expressive vector pBV220 NT4 TAT 6×His VHLβ was constructed by a routine molecular biological method. These results lay a foundation for further research of fusion gene to treat RCC.