酵母双杂交系统筛选IEX-1相互作用蛋白的研究
摘要
[目的]
通过酵母双杂交技术筛选成人肝cDNA文库,寻找IEX-1相互作用蛋白,为进一步深入功能机制研究奠定基础。
[材料与方法]
应用酵母双杂交技术,通过筛选成人肝cDNA文库,寻找IEX-1相互作用蛋白。用Trizol方法提取骨肉瘤MG-63细胞RNA, RT-PCR扩增IEX-1基因编码区,电泳回收Spel/NotI双酶切连入pDBLeu载体,构建表达质粒。进一步进行,诱饵蛋白的自激活检测,文库筛选和回转验证等实验获得真阳性克隆。提取真阳性的克隆中cDNA融合质粒,测序,进一步生物信息分析。
[结果]
1.在融合蛋白表达质粒的构建过程中,我们成功获得IEX-1基因编码区片段并构建出融合蛋白表达质粒pDBLeu-IEX-1,经过酶切鉴定后测序证实各诱饵载体均切出了正常长度的插入片段和载体骨架片段。
2.将重组成功的pDBLeu-IEX-1和空载体pDBLeu转化入酵母菌AH109进行毒性及自激活检测,结果发现pDBLeu- IEX-1对酵母菌AH109无毒性作用及自激活作用。
3.使用诱饵质粒pDBLeu-IEX-1转化感受态细胞,30℃培养3～8 d后,将菌液涂布于SD/-Trp/-Leu/-His/-Ade平板上初步筛选阳性克隆,初筛的阳性克隆进行X-α-gal显色进一步确认,结果共筛选出12个阳性克隆。
4.提取12个克隆中cDNA融合质粒,与pDBLeu-IEX-1共转化入酵母菌株AH109回转验证,结果表明其中4个克隆为真阳性克隆,其他为假阳性克隆。将这4个真阳性克隆扩大培养、提质粒后测序,查出该cDNA融合质粒对应的基因,最终确定四个IEX-1相互作用的蛋白：C reactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1)。
[结论]：
1.成功构建酵母双杂交系统,经检验能够进行进一步的IEX-1相互作用的蛋白的筛选；
2.通过酵母双杂交系统筛选出四个IEX-1相互作用的蛋白：Creactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1)。为进一步深入骨肉瘤发病机制研究奠定基础。
OBJECTIVE:
By screening of adult liver cDNA library use yeast two-hybrid to look for IEX-1 interaction protein and prepare for the further study of its functional mechanism.
MATERIALS AND METHODS:
Look for IEX-1 interaction protein through the screening of adult liver cDNA library using yeast two-hybrid technology. Extracted RNA from osteosarcoma MG-63 cells through Trizol method.RT-PCR amplified IEX-1 gene coding region, then through electrophoresis retrieved the amplified IEX-1 gene coding region and after SpeI/NotI double digestion they were connected with pDBLeu vector, thus expression plasmids were constructed. After detecting self-activation of bait proteins, screening Library and verification of rotary experiments we acquired true positive clones. Extracted the true positive fusion plasmid cDNA clones, sequenced and performed further analysis of biological information.
RESUITS:
1. During the Construction of fusion protein expression plasmid, we successfully acquired IEX-1 gene coding region fragments and constructed fusion protein expression plasmids pDBLeu-IEX-1,which We confirmed through restriction enzyme digestion and sequencing were cut out of the normal length inserts and vector backbone fragments.
2. Transformed the recombinant pDBLeu-IEX-1 that was successfully constructed and empty vector pDBLeu into yeast AH109 to perform its toxicity and self-activation test. Then We found that there was no toxic effects for pDBLeu-IEX-1 to yeast AH109 and self-activation.
3. Used the bait plasmid pDBLeu-IEX-1 to transform competent cells, and cultured them at the temperature of 30℃for 3～8 days,then coated the bacteria on SD/-Trp/-Leu/-His/-Ade plate to select positive clones and screened the positive clones through X-a-gal color for further confirming. The results show that 12 positive clones were selected.
4. Extracted the fusion plasmid of 12 cDNA clones, and transformed them together with pDBLeu-IEX-1 into yeast strain AH109 for rotary validation. Results showed that four clones of them were the true positive clones and the others were the false positive clones. Expanded and trained these four true positive clones and extracted plasmid for sequencing to identify the corresponding gene of the fusion plasmid cDNA. Finally four IEX-1 interaction proteins were determined:C reactive protein (CRP), Clusterin (CLU), Cathepsin B (CTSB), Heat shock protein 105 (HSPH1).
CONCIUSION:
1. Yeast two-hybrid system was successfully constructed and by the test it could be used for the further screening of IEX-1 interaction protein;
2. Using yeast two-hybrid system We screened out four IEX-1 interaction protein:C reactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1). Our experiment lays the foundation for the further pathogenesis study of osteosarcoma.
通过酵母双杂交技术筛选成人肝cDNA文库,寻找IEX-1相互作用蛋白,为进一步深入功能机制研究奠定基础。
[材料与方法]
应用酵母双杂交技术,通过筛选成人肝cDNA文库,寻找IEX-1相互作用蛋白。用Trizol方法提取骨肉瘤MG-63细胞RNA, RT-PCR扩增IEX-1基因编码区,电泳回收Spel/NotI双酶切连入pDBLeu载体,构建表达质粒。进一步进行,诱饵蛋白的自激活检测,文库筛选和回转验证等实验获得真阳性克隆。提取真阳性的克隆中cDNA融合质粒,测序,进一步生物信息分析。
[结果]
1.在融合蛋白表达质粒的构建过程中,我们成功获得IEX-1基因编码区片段并构建出融合蛋白表达质粒pDBLeu-IEX-1,经过酶切鉴定后测序证实各诱饵载体均切出了正常长度的插入片段和载体骨架片段。
2.将重组成功的pDBLeu-IEX-1和空载体pDBLeu转化入酵母菌AH109进行毒性及自激活检测,结果发现pDBLeu- IEX-1对酵母菌AH109无毒性作用及自激活作用。
3.使用诱饵质粒pDBLeu-IEX-1转化感受态细胞,30℃培养3～8 d后,将菌液涂布于SD/-Trp/-Leu/-His/-Ade平板上初步筛选阳性克隆,初筛的阳性克隆进行X-α-gal显色进一步确认,结果共筛选出12个阳性克隆。
4.提取12个克隆中cDNA融合质粒,与pDBLeu-IEX-1共转化入酵母菌株AH109回转验证,结果表明其中4个克隆为真阳性克隆,其他为假阳性克隆。将这4个真阳性克隆扩大培养、提质粒后测序,查出该cDNA融合质粒对应的基因,最终确定四个IEX-1相互作用的蛋白：C reactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1)。
[结论]：
1.成功构建酵母双杂交系统,经检验能够进行进一步的IEX-1相互作用的蛋白的筛选；
2.通过酵母双杂交系统筛选出四个IEX-1相互作用的蛋白：Creactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1)。为进一步深入骨肉瘤发病机制研究奠定基础。
OBJECTIVE:
By screening of adult liver cDNA library use yeast two-hybrid to look for IEX-1 interaction protein and prepare for the further study of its functional mechanism.
MATERIALS AND METHODS:
Look for IEX-1 interaction protein through the screening of adult liver cDNA library using yeast two-hybrid technology. Extracted RNA from osteosarcoma MG-63 cells through Trizol method.RT-PCR amplified IEX-1 gene coding region, then through electrophoresis retrieved the amplified IEX-1 gene coding region and after SpeI/NotI double digestion they were connected with pDBLeu vector, thus expression plasmids were constructed. After detecting self-activation of bait proteins, screening Library and verification of rotary experiments we acquired true positive clones. Extracted the true positive fusion plasmid cDNA clones, sequenced and performed further analysis of biological information.
RESUITS:
1. During the Construction of fusion protein expression plasmid, we successfully acquired IEX-1 gene coding region fragments and constructed fusion protein expression plasmids pDBLeu-IEX-1,which We confirmed through restriction enzyme digestion and sequencing were cut out of the normal length inserts and vector backbone fragments.
2. Transformed the recombinant pDBLeu-IEX-1 that was successfully constructed and empty vector pDBLeu into yeast AH109 to perform its toxicity and self-activation test. Then We found that there was no toxic effects for pDBLeu-IEX-1 to yeast AH109 and self-activation.
3. Used the bait plasmid pDBLeu-IEX-1 to transform competent cells, and cultured them at the temperature of 30℃for 3～8 days,then coated the bacteria on SD/-Trp/-Leu/-His/-Ade plate to select positive clones and screened the positive clones through X-a-gal color for further confirming. The results show that 12 positive clones were selected.
4. Extracted the fusion plasmid of 12 cDNA clones, and transformed them together with pDBLeu-IEX-1 into yeast strain AH109 for rotary validation. Results showed that four clones of them were the true positive clones and the others were the false positive clones. Expanded and trained these four true positive clones and extracted plasmid for sequencing to identify the corresponding gene of the fusion plasmid cDNA. Finally four IEX-1 interaction proteins were determined:C reactive protein (CRP), Clusterin (CLU), Cathepsin B (CTSB), Heat shock protein 105 (HSPH1).
CONCIUSION:
1. Yeast two-hybrid system was successfully constructed and by the test it could be used for the further screening of IEX-1 interaction protein;
2. Using yeast two-hybrid system We screened out four IEX-1 interaction protein:C reactive protein (CRP)、Clusterin (CLU)、Cathepsin B (CTSB)、Heat shock protein 105 (HSPH1). Our experiment lays the foundation for the further pathogenesis study of osteosarcoma.
引文
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