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左魁阳, 陈梦茜, 韩瑞杰, 王小花, 孟娜娜, 金秀东, 刘海峰. TGF-1和TGF-Ⅱ型受体酵母表达载体构建[J]. 中国公共卫生, 2017, 33(6): 926-929. DOI: 10.11847/zgggws2017-33-06-15
引用本文: 左魁阳, 陈梦茜, 韩瑞杰, 王小花, 孟娜娜, 金秀东, 刘海峰. TGF-1和TGF-Ⅱ型受体酵母表达载体构建[J]. 中国公共卫生, 2017, 33(6): 926-929. DOI: 10.11847/zgggws2017-33-06-15
ZUO Kui-yang, CHEN Meng-xi, HAN Rui-jie.et al, . 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[J]. Chinese Journal of Public Health, 2017, 33(6): 926-929. DOI: 10.11847/zgggws2017-33-06-15
Citation: ZUO Kui-yang, CHEN Meng-xi, HAN Rui-jie.et al, . 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[J]. Chinese Journal of Public Health, 2017, 33(6): 926-929. DOI: 10.11847/zgggws2017-33-06-15

TGF-1和TGF-Ⅱ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

  • 摘要: 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cDNA,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pGBKT7和pGADT7;经过PCR扩增、双酶切和DNA测序鉴定后,构建质粒pGBKT7-TGF-β1和pGADT7-TβRⅡ-A、pGADT7-TβRⅡ-B、pGADT7-TβRⅡ-C和pGADT7-TβRⅡ-D。结果 成功扩增TGF-β1基因(1 200 bp)和TβRⅡ-A(453 bp)、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pGBKT7-TGF-β1和pGADT7-TβRⅡ-A、pGADT7-TβRⅡ-B、pGADT7-TβRⅡ-C和pGADT7-TβRⅡ-D酵母双杂交表达载体。

     

    Abstract: 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.

     

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