酵母双杂交技术筛选人95D细胞中肺癌转移相关蛋白1相互作用蛋白及其验证

 目的 通过酵母双杂交实验、免疫共沉淀及GST pull down技术筛选并验证肺癌细胞系95D细胞中与肺癌转移相关蛋白1(lung cancer metastasis-related protein 1,LCMR1)相互作用蛋白,为进一步研究LCMR1在肺癌发生发展中的作用提供科学依据。方法 将诱饵质粒pGBKT7-LCMR转化AH109,应用酵母双杂交系统筛选出95D细胞中与LCMR1相互作用的候选克隆;应用反复划线法、X-α-Gal显色法和共转化回复验证法排除假阳性克隆,对真阳性克隆进行测序及生物学分析;构建含Myc标签的LCMR1融合蛋白的重组载体pCMV - Myc-LCMR1载体,以及带flag标签的目标相互作用蛋白载体,共转染细胞,利用免疫共沉淀技术验证LCMR1与相互作用蛋白之前的相互作用;融合蛋白沉降(GST pull- down)法进一步体外验证LCMR1与相互作用蛋白之间的作用。结果 诱饵质粒pGBKT7 -LCMR1与95D细胞cDNA文库共转化AH109,初步获得20个候选克隆;重复划线法后获得16个阳性克隆,X-α-Gal显色法获得12个蓝色阳性克隆,进一步将文库质粒与诱饵质粒共转化酵母菌回复验证,最终获得6个真阳性克隆;成功构建含标签重组载pCMV-Myc-LCMR1、pcDNA3.0-flag-RPL29及pcDNA3.0- flag-GNG5载体,经免疫共沉淀验证LCMR1与RPL29在细胞内有相互作用,与GNG5在细胞内无相互作用;GST pull-down实验验证LCMR1与RPL29在体外有相互作用,与GNG5在细胞外无相互作用。结论 酵母双杂交技术筛选出95D细胞中相互作用蛋白6个,经免疫共沉淀和GST pull-down实验验证LCMR1与RPL29体内体外均有相互作用。

Screening and identification of candidate proteins interacting with lung cancer metastasis-related protein 1 of human 95D cell line cDNA library by yeast two hybridization

Objective To screen and identify the proteins that interact with LCMR1 from human 95D cell line library by the yeast two-hybrid system.Methods The bait plasmid, pGBKT7-LCMR1 , which expressed LCMR1 fusion protein, was transformed into AH109 yeast strains before yeast two-hybrid screening was performed by mating AH109 with Y187 containing human 95D cell cDNA library plasmids. SD/-Ade/-His/-Leu/-Trp medium and X-α-Gal were used for selecting and screening the interacting proteins.Moreover, back-cross was performed to confirm the positive clones .The vectors of Myc-tagged fusion protein and flag-tagged interacting proteins were constructed, identified and cotransfected into human embryo kidney 293 cells.The interactions between them were investigated by the immunoprecipitation(CO-IP) and GST pull-down system.Results The bait plasmid pGBKT7-LCMR1 was identified and transformed into AH109 and six true positive proteins with known functions were obtained through sequencing and bioinformatics analysis, which were GNG5, RPL29, DEK, CCAR1, G3BP1 and LGMN. GNG5 and RPL29 were selected for further study. The eukaryotic expression vectors of pcDNA3.0-flag -RPL29, pcDNA3.0 -flag-GNG5 and pCMV-Myc-LCMR1 were constructed and confirmed with double restriction enzyme digestion and co-transfected into 293 cell line.The interactions between RPL29 and LCMR1 were identified with Western blot after Co-IP, andno interactions between GNG5 and LCMR1 were detected.The same results were found in the subsequent experiment of GST pull down.Conclusions Proteins in 95D cell line library that can interact with LCMR1 are successfully screened out and identified.The interactions between LCMR1 and RPL29 are confirmed by the CO-IP and GST pull-down system.