细菌双杂交系统在细胞外蛋白质相互作用中的应用

Application of a bacterial two-hybrid system in extracellular protein interactions

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 the extracellular protein-protein interactions using a bacterial two-hybrid system. Methods: Using Stratagene BacterioMatch two-hybrid system, a fused expression plasmid was constructed with pTRG and TRAIL'S soluble extracellular domain as the 'bait', while the fused expression plasmids were also constructed with pBT and extracellular domain of DR4, DR5 or OPG as the 'target'. Each recombinant pTRG and pBT were co-transfor-mated into bacterioMatch two-hybrid system reporter strain E. coli XL1-Blue MRF. The protein-protein interactions were detected by its carbenicillin-resistant property and validated by β-galactosidase activation. Results: Four groups, namely pTRG-TRAIL and pBT-DR4,pTRG-TRAIL and pBT-DR5, controls (pBT-LGF2 and PTRG-Gal11) ,and pTRG-TRAIL and pBT-OPG, grew as carbenicillin-resistant colonies. The first 3 groups showed higher resistance to carbenicillin (above 500 μg/ml) and the last one showed lower resistance (under 250 ng/ml). The positive colonies were further validated by β-galactosidase activation on X-Gal-PETG plates. The others, including pTRG-TRAIL and pBT-LGF2, pBT-DR4 or pBT-DR5 or pBT-OPG and pTRG-Gal11, or non-co-transformed plasmids themselves, were negative on carbenicillin or X-Gal-PETG plates. Conclusion: Bacterial two-hybrid system can be used for analysis and identification of certain extracellular protein-protein interactions.