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目的:探索应用细菌双杂交系统在细胞外蛋白质之间相互作用的可行性。方法:应用Stratagene BacterioMatch two hy-brid system,以pTRG构建编码TRAIL细胞外可溶性区域序列融合表达质粒;以pBT构建编码DR4、DR5、OPG等的细胞外区域序列融合表达质粒。重组质粒分别相应共转化报告菌株XL1-Blue MRF,报道基因产物以抗羧苄青霉素和β-半乳糖苷酶活性作为转录激活的指标。结果:pTRG-TRAIL与pBT-DR4,pTRG-TRAIL与pBT-DR5及对照组分别共转化后在高浓度羧苄青霉素(500μg/ml以上)筛选下宿主菌呈转录激活状态,pTRG-TRAIL与pBT-OPG组共转化后在低浓度羧苄青霉素(250μg/ml以下)筛选下宿主菌呈转录激活状态,用X-Gal-PETG平皿可以进一步验证阳性克隆。对照组pTRG-TRAIL与pBT-LGF2,pBT-DR4、pBT-DR5或pBT-OPG与pTRG-Gal114组共转化后呈阴性。结论:所用的双杂交序列皆为编码这些蛋白质的细胞外区域,而这些蛋白质本身的相互作用已有其他报道验证,所以细菌双杂交系统可以用于细胞外蛋白质的相互作用研究。
Objective: To explore the feasibility of using bacterial two-hybrid system to interact with extracellular proteins. Methods: The fusion plasmid pCRG encoding the soluble region of TRAIL was constructed by Stratagene BacterioMatch two hy-brid system. The pBT was used to construct the fusion expression plasmid encoding extracellular region of DR4, DR5 and OPG. The recombinant plasmids were co-transformed respectively with the reporter strain XL1-Blue MRF, and the reporter gene products were used as indicators of transcriptional activation of carbenicillin and β-galactosidase activity. Results: After co-transformation of pTRG-TRAIL with pBT-DR4, pTRG-TRAIL and pBT-DR5 and the control group, the host bacteria were transcriptionally activated by high concentration of carbenicillin (500μg / ml or more) -OPG group, the host bacteria were transcribed and activated under the screening of low concentration of carbenicillin (250μg / ml or less). The positive clones could be further verified by X-Gal-PETG plate. The control group pTRG-TRAIL and pBT-LGF2, pBT-DR4, pBT-DR5 or pBT-OPG and pTRG-Gal114 co-transformation was negative. CONCLUSION: The two-hybrid sequences used are all extracellular regions that encode these proteins. The interaction of these proteins has been reported in other reports. Therefore, the bacterial two-hybrid system can be used to study the interaction of extracellular proteins.