与甲型副伤寒杆菌噬菌体LSPA1早期蛋白GP23相互作用的宿主蛋白的筛选  

Screening of host protein interacting with early protein GP23 of bacteriophage LSPA1 of Salmonella Paratyphi A

作　　者：毛普加 冯梦蝶[1] 洪愉[3] 许泽仰 赵继华[3] 杨洪文[3] 宋武战[3] 王纪爱[2] 饶贤才[4] 黄芬[1] 井申荣[1] 曾韦锟[1,2] 

机构地区：[1]昆明理工大学医学院病原微生物学实验室,云南昆明650500 [2]昆明学院医学院临床检验教研室,云南昆明650214 [3]成都军区昆明总医院核医学科,云南昆明650532 [4]第三军医大学微生物学教研室,重庆404100

出　　处：《山东大学学报（医学版）》2015年第7期29-33,共5页Journal of Shandong University:Health Sciences

基　　金：云南省应用基础研究面上项目(2012FB135);国家自然科学基金(31160193)

摘　　要：目的筛选与甲型副伤寒杆菌（副甲）CMCC50973噬菌体LSPA1早期蛋白GP23相互作用的宿主蛋白。方法 Sau3AⅠ酶切副甲基因组,回收250-1 500 bp的条带与p AT质粒连接,构建p AT-副甲基因文库;扩增噬菌体早期基因gp23,与p BT构建p BT-gp23作为诱饵质粒;将p AT-副甲基因文库和p BT-gp23共转KS宿主菌,利用细菌双杂交技术筛选蓝色单菌。检测筛选出阳性单菌的Lac Z活性,并提质粒测序,分析其编码蛋白。结果成功构建副甲基因文库,其库容量满足实验的要求,基因文库阳性率接近100%;筛选出的插入基因为编码嘌呤透性酶。结论宿主蛋白（嘌呤透性酶）可能与副甲噬菌体LSPA1早期蛋白GP23有相互作用。Objective To screen the host protein interacting with early protein GP23 of bacteriophage LSPA1 of Salmonella Paratyphi A（ S. Paratyphi A） CM CC50973. Methods The genome of S. Paratyphi A（ CM CC50973） was digested by Sau3AⅠ and the recovered bands（ 250 to 1 500 bp） were ligated with p AT plasmid to create the S. Paratyphi A genomic library. gp23,an early gene of phage LSPA1 genome,was ligated with p BT as a bait plasmid（ p BT-gp23）.The S. Paratyphi A genomic library plasmids and p BT-gp23 were co-transformed into the host bacteria KS. Furthermore,the bacterial two-hybrid was used to screen blue single bacterium. Positive clones were tested by the activity of Lac Z.Positive plasmids were extracted,sequenced and analyzed. Results S. Paratyphi A genomic library was successfullyconstructed and its capatity met the requirements of the experiment. Positive rate of the library was about 100%. The gene of positive clone was putative purine permease by sequencing and BLAST. Conclusion Host protein（ putative purine permease） may interact with early protein GP23 of bacteriophage LSPA1 of S. Paratyphi A.

关 键 词：甲型副伤寒杆菌 噬菌体 细菌双杂交 

分 类 号：Q939.47[生物学—微生物学] R378.2[医药卫生—病原生物学]