TGF-β1和TGF-βⅡ型受体酵母表达载体构建

Construction and identification of yeast two-hybrid expression vector containing human transforming growth factor-beta 1 and different fragments of transforming growth factor-beta receptor type Ⅱ

目的克隆人转化生长因子β1(TGF-β1)和4个受体基因片段,构建酵母表达载体,为应用酵母双杂交筛选与TGF-β1相互作用的TβRⅡ区域提供技术支持。方法提取人血液中RNA,逆转录转为c DNA,PCR扩增TGF-β1和4个受体(TβRⅡ-A、TβRⅡ-B、TβRⅡ-C、TβRⅡ-D)基因片段,双酶切后,分别插入酵母双杂交表达载体p GBKT7和p GADT7;经过PCR扩增、双酶切和DNA测序鉴定后,构建质粒p GBKT7-TGF-β1和p GADT7-TβRⅡ-A、p GADT7-TβRⅡ-B、p GADT7-TβRⅡ-C和p GADT7-TβRⅡ-D。结果成功扩增TGF-β1基因(1 200 bp)和TβRⅡ-A(453bp)、TβRⅡ-B(366 bp)、TβRⅡ-C(276 bp)和TβRⅡ-D(165 bp)基因片段,经PCR、双酶切和DNA测序鉴定插入到酵母双杂交载体序列正确。结论成功构建p GBKT7-TGF-β1和p GADT7-TβRⅡ-A、p GADT7-TβRⅡ-B、p GADT7-TβRⅡC和p GADT7-TβRⅡ-D酵母双杂交表达载体。

Objective To clone human transforming growth factor-beta 1 （TGF-β1） and 4 genome fragments of its receptor and to construct the yeast expression vector to provide technical support for using yeast two-hybrid in identification of interaction domain of TGF-β type Ⅱ receptor （TβRⅡ） with TGF-β1.Methods The gene TGF-β1 and gene fragments of TβRⅡ from the RNA of human peripheral blood were amplified with regular PCR using specific primers,and then cloned into yeast two-hybrid expression vector pGBKT7 and pGADT7.The recombinant plasmids pGBKT7-TGF-β1 and pGADT7-TβRⅡ-A,pGADT7-TβRⅡ-B,pGADT7-TβRⅡ-C,pGADT7-TβRⅡ-D sequences were confirmed with PCR,restriction endonucleases digestion,and automated DNA sequencing.Results The yeast two-hybrid expression vector pGBKT7-TGF-β1 （1 200 bp） and pGADT7-TβRⅡ-A （453 bp）,pGADT7-TβRⅡ-B （366 bp）,pGADT7-TβRⅡ-C （276 bp）,pGADT7-TβRⅡ-D （165 bp） were successfully constructed and the sequences of the plasmids’ DNA were correct.Conclusion The yeast two-hybrid expression vectors pGBKTT-TGFβ1 and pGADT7-TβRⅡA,B,C,and D were successfully constructed.