活细胞内筛选地塞米松衍生物作用靶蛋白的研究
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摘要
第一部分
糖皮质激素受体结合域诱饵表达载体的构建和鉴定
目的构建糖皮质激素受体α配体结合域(GRα-LBD)的诱饵表达载体,为小分子配体酵母三杂交系统的建立奠定基础。
方法RT-PCR扩增K562细胞的GRα-LBD,克隆入诱饵载体pGBKT7中,测序正确后,再把构建好的诱饵载体pGBKT7-GRα-LBD转化到酵母AH109细胞中,提取酵母蛋白,并用Western blot分析诱饵蛋白的表达情况。同时检测诱饵蛋白的毒性、渗漏和自激活作用。
结果成功扩增了GRα-LBD,并分别成功克隆到pGBKT7中,测序结果正确。诱饵载体成功转化到酵母AH109细胞中,无毒性、渗漏和自激活作用,Western blot分析也证实了酵母细胞表达诱饵蛋白。
结论成功构建了GRα-LBD酵母诱饵表达载体,为建立小分子配体酵母三杂交系统奠定了基础。
第二部分
人K562细胞cDNA文库的扩增、纯化、鉴定和酵母细胞转化
目的对预转化到大肠DH5α中的人K562细胞cDNA文库进行扩增,纯化和鉴定并将文库质粒转化酵母为下一步的靶蛋白的筛选做准备。
方法对K562细胞cDNA文库进行扩增,提取文库质粒,将文库质粒转化酵母Y187细胞,并用PCR和酶切鉴定转化结果。
结果成功的扩增人K562细胞cDNA文库,并验证了文库的多样性,将文库质粒成功转化酵母Y187细胞。
结论K562细胞cDNA文库的成功扩增,纯化,鉴定,为进一步的文库筛选奠定了基础。
第三部分
酵母三杂交技术筛选地塞米松衍生物作用的靶蛋白
目的通过酵母三杂交技术在人K562细胞cDNA中筛选地塞米松衍生物作用的靶蛋白。
方法通过将转化有诱饵质粒pGBKT7-GRα-LBD的酵母AH109细胞、转化有文库质粒的酵母Y187细胞和终浓度为0.4μg/ml的地塞米松衍生物三种物质放到一起交配后,将交配后的细胞铺于SD/-Trp/-Leu/-His的缺陷培养基上,然后将SD/-Trp/-Leu/-His培养基上生长的克隆挑取到SD/-Trp/-Leu/-His/-Ade /X-α-gal的缺陷培养基上进行营养缺陷筛选,重复挑取三次后仍为蓝色的克隆可能是阳性克隆,然后提取酵母质粒并转化大肠杆菌DH5α感受态细胞,提取大肠杆菌DH5α中的质粒,并通过对所提取的所有文库质粒进行PCR扩增和对PCR产物的单酶切分析,将文库质粒归类,归类后从每一类中随机选择一个代表,通过酵母回转实验进行验证。酵母回转实验阳性的质粒送上海生工测序,通过对测序结果的比对和进一步的分析来确定所筛选到的靶蛋白的特征。
结果共测序43个,其中有6个是重复的,实际上我们共筛选到37个不同的蛋白,经过回转实验验证,证明20个是阳性克隆,其中一个是新蛋白。从中选择6个进行一对一的酵母交配实验后,发现DDX28和RanBP9/RanBPM是与地塞米松衍生物相互作用的靶蛋白。
结论本研究我们筛选到了2个与地塞米松衍生物相互作用的靶蛋白,分别是DDX28和RanBP9/RanBPM。这将为地塞米松衍生物抗肿瘤的分子机制研究提供线索。
PART1
CONSTRUCTION AND IDENTIFICATION OF THE BAIT EXPRESSION VECTOR OF GLUCOCORTICOID RECEPTOR BINDING DOMAIN
Objective: To construct the bait expression plasmid pGBKT7-GRα-LBD of glucocorticoid receptor 1igand binding domain, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.
Methods: The fragments of GRα-LBD of human K562 cell were amplified by RT-PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vector pGBKT7-GRα-LBD was transformed into AH109 yeast cells. And the expression of the bait protein was analyzed by western blot. Toxicity,leakage and self-activation of the bait protein were detected simultaneously. Results: GRα-LBD was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 yeast cells successfully and no toxicity, leakage and self-activation were found.The expression of the bait protein was confirmed by western blot.
Conclusion: The bait expression vector pGBKT7-GRα-LBD was constructed successfully, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.
PART2
AMPLI FICATION, PURIFICATION, IDENTIFICATION OF HUMAN K562 CELL cDNA LIBRARY AND ITS TRANSFORMATION INTO YEAST CELL
Objective: To ampliy,purify and identify the cDNA library of K562 cell in Eoli DH5αand to transform it into yeast cell for subsequent target protein screening.
Methods: The cDNA library of human K562 cell was amplified.Then the library plasmids were extracted and transformed into Y187 yeast cell.The results of transformation were verified by PCR and restriction enzyme analysis.
Results: The cDNA library of human K562 cell was successfully amplified and the diversities were verificated. The cDNA library of human K562 cell was also successfully transformed into Y187 yeast cell.
Conclusion: Successfully amplification,purification and identification of the cDNA library of human K562 cell which lay the foundations for subsequent protein screening.
PART3
SCREENING TARGET PROTEINS OF DEXAMETHASONE DERIVATE BY YEAST THREE-HYBRID SYSTEM
Objective: Screening target proteins of dexamethasone derivate in K562 cell cDNA library by yeast three-hybrid system.
Methods: Yeast strain AH109 containing bait plasmid pGBKT7-GRα-LBD, yeast strain Y187 transformed by K562 cell cDNA library and 0.4μg/ml dexamethasone derivate were mated together,spread the mating product to SD/-Trp/-Leu/-His dropout plate, then pick clones from SD/-Trp/-Leu/-His dropout plate to SD/-Trp/-Leu/-His/-Ade/X-α-gal dropout plate for further nutrition dropout screening.Blue clones were presumed positive clones after 3 times nutrition dropout screening.Yeast plasmids were extracted and transformed into Ecoi DH5α,subsequently plasmids of Ecoi DH5αwere extracted and library fragments were amplifed by PCR and amplification products were digested by only one restrict enzyme. Library plasmids were sorted by PCR and restrict enzyme analysis. We chosen one from every sort randomly as representation to verity weather it was really positive by yeast retransformation test. Positive plasmids were analysised by DNA sequencing. Target protein property was described by analysising sequencing results subsequently.
Results: We sequenced forty-three clones at all, and six clones were duplication of the total, in fact we screened thirty-seven different proteins in our experiment. Yeast retransformation test showed that there were twenty politive clones and one new protein among these clones. Yeast mating test were performed of six clones from those that we had screened. Only DDX28 and RanBP9/RanBPM were target proteins interacting with dexamethasone derivate.
Conclusion: We screened two target proteins(DDX28 and RanBP9/RanBPM) interacting with dexamethasone derivate. It may be as one envidence on study of dexamethasone derivate anticancer molecular mechanism.
糖皮质激素受体结合域诱饵表达载体的构建和鉴定
目的构建糖皮质激素受体α配体结合域(GRα-LBD)的诱饵表达载体,为小分子配体酵母三杂交系统的建立奠定基础。
方法RT-PCR扩增K562细胞的GRα-LBD,克隆入诱饵载体pGBKT7中,测序正确后,再把构建好的诱饵载体pGBKT7-GRα-LBD转化到酵母AH109细胞中,提取酵母蛋白,并用Western blot分析诱饵蛋白的表达情况。同时检测诱饵蛋白的毒性、渗漏和自激活作用。
结果成功扩增了GRα-LBD,并分别成功克隆到pGBKT7中,测序结果正确。诱饵载体成功转化到酵母AH109细胞中,无毒性、渗漏和自激活作用,Western blot分析也证实了酵母细胞表达诱饵蛋白。
结论成功构建了GRα-LBD酵母诱饵表达载体,为建立小分子配体酵母三杂交系统奠定了基础。
第二部分
人K562细胞cDNA文库的扩增、纯化、鉴定和酵母细胞转化
目的对预转化到大肠DH5α中的人K562细胞cDNA文库进行扩增,纯化和鉴定并将文库质粒转化酵母为下一步的靶蛋白的筛选做准备。
方法对K562细胞cDNA文库进行扩增,提取文库质粒,将文库质粒转化酵母Y187细胞,并用PCR和酶切鉴定转化结果。
结果成功的扩增人K562细胞cDNA文库,并验证了文库的多样性,将文库质粒成功转化酵母Y187细胞。
结论K562细胞cDNA文库的成功扩增,纯化,鉴定,为进一步的文库筛选奠定了基础。
第三部分
酵母三杂交技术筛选地塞米松衍生物作用的靶蛋白
目的通过酵母三杂交技术在人K562细胞cDNA中筛选地塞米松衍生物作用的靶蛋白。
方法通过将转化有诱饵质粒pGBKT7-GRα-LBD的酵母AH109细胞、转化有文库质粒的酵母Y187细胞和终浓度为0.4μg/ml的地塞米松衍生物三种物质放到一起交配后,将交配后的细胞铺于SD/-Trp/-Leu/-His的缺陷培养基上,然后将SD/-Trp/-Leu/-His培养基上生长的克隆挑取到SD/-Trp/-Leu/-His/-Ade /X-α-gal的缺陷培养基上进行营养缺陷筛选,重复挑取三次后仍为蓝色的克隆可能是阳性克隆,然后提取酵母质粒并转化大肠杆菌DH5α感受态细胞,提取大肠杆菌DH5α中的质粒,并通过对所提取的所有文库质粒进行PCR扩增和对PCR产物的单酶切分析,将文库质粒归类,归类后从每一类中随机选择一个代表,通过酵母回转实验进行验证。酵母回转实验阳性的质粒送上海生工测序,通过对测序结果的比对和进一步的分析来确定所筛选到的靶蛋白的特征。
结果共测序43个,其中有6个是重复的,实际上我们共筛选到37个不同的蛋白,经过回转实验验证,证明20个是阳性克隆,其中一个是新蛋白。从中选择6个进行一对一的酵母交配实验后,发现DDX28和RanBP9/RanBPM是与地塞米松衍生物相互作用的靶蛋白。
结论本研究我们筛选到了2个与地塞米松衍生物相互作用的靶蛋白,分别是DDX28和RanBP9/RanBPM。这将为地塞米松衍生物抗肿瘤的分子机制研究提供线索。
PART1
CONSTRUCTION AND IDENTIFICATION OF THE BAIT EXPRESSION VECTOR OF GLUCOCORTICOID RECEPTOR BINDING DOMAIN
Objective: To construct the bait expression plasmid pGBKT7-GRα-LBD of glucocorticoid receptor 1igand binding domain, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.
Methods: The fragments of GRα-LBD of human K562 cell were amplified by RT-PCR, and then were cloned into the bait expression vector pGBKT7. After being verified by sequencing, the bait vector pGBKT7-GRα-LBD was transformed into AH109 yeast cells. And the expression of the bait protein was analyzed by western blot. Toxicity,leakage and self-activation of the bait protein were detected simultaneously. Results: GRα-LBD was amplified and cloned into pGBKT7 successfully. The bait vector was transformed into AH109 yeast cells successfully and no toxicity, leakage and self-activation were found.The expression of the bait protein was confirmed by western blot.
Conclusion: The bait expression vector pGBKT7-GRα-LBD was constructed successfully, which lay the foundations for constructing small molecule ligand yeast three-hybrid system.
PART2
AMPLI FICATION, PURIFICATION, IDENTIFICATION OF HUMAN K562 CELL cDNA LIBRARY AND ITS TRANSFORMATION INTO YEAST CELL
Objective: To ampliy,purify and identify the cDNA library of K562 cell in Eoli DH5αand to transform it into yeast cell for subsequent target protein screening.
Methods: The cDNA library of human K562 cell was amplified.Then the library plasmids were extracted and transformed into Y187 yeast cell.The results of transformation were verified by PCR and restriction enzyme analysis.
Results: The cDNA library of human K562 cell was successfully amplified and the diversities were verificated. The cDNA library of human K562 cell was also successfully transformed into Y187 yeast cell.
Conclusion: Successfully amplification,purification and identification of the cDNA library of human K562 cell which lay the foundations for subsequent protein screening.
PART3
SCREENING TARGET PROTEINS OF DEXAMETHASONE DERIVATE BY YEAST THREE-HYBRID SYSTEM
Objective: Screening target proteins of dexamethasone derivate in K562 cell cDNA library by yeast three-hybrid system.
Methods: Yeast strain AH109 containing bait plasmid pGBKT7-GRα-LBD, yeast strain Y187 transformed by K562 cell cDNA library and 0.4μg/ml dexamethasone derivate were mated together,spread the mating product to SD/-Trp/-Leu/-His dropout plate, then pick clones from SD/-Trp/-Leu/-His dropout plate to SD/-Trp/-Leu/-His/-Ade/X-α-gal dropout plate for further nutrition dropout screening.Blue clones were presumed positive clones after 3 times nutrition dropout screening.Yeast plasmids were extracted and transformed into Ecoi DH5α,subsequently plasmids of Ecoi DH5αwere extracted and library fragments were amplifed by PCR and amplification products were digested by only one restrict enzyme. Library plasmids were sorted by PCR and restrict enzyme analysis. We chosen one from every sort randomly as representation to verity weather it was really positive by yeast retransformation test. Positive plasmids were analysised by DNA sequencing. Target protein property was described by analysising sequencing results subsequently.
Results: We sequenced forty-three clones at all, and six clones were duplication of the total, in fact we screened thirty-seven different proteins in our experiment. Yeast retransformation test showed that there were twenty politive clones and one new protein among these clones. Yeast mating test were performed of six clones from those that we had screened. Only DDX28 and RanBP9/RanBPM were target proteins interacting with dexamethasone derivate.
Conclusion: We screened two target proteins(DDX28 and RanBP9/RanBPM) interacting with dexamethasone derivate. It may be as one envidence on study of dexamethasone derivate anticancer molecular mechanism.
引文
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