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

doi:10.6040/j.issn.1671-7554.0.2014.903

山东大学学报（医学版） ›› 2015, Vol. 53 ›› Issue (7): 29-33.doi: 10.6040/j.issn.1671-7554.0.2014.903

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

毛普加^1,2, 冯梦蝶¹, 洪愉³, 许泽仰¹, 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

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

收稿日期:2014-12-05 修回日期:2015-03-23 出版日期:2015-07-10 发布日期:2015-07-10
通讯作者: 曾韦锟。E-mail:zengweikun@gmail.com E-mail:zengweikun@gmail.com
基金资助:
云南省应用基础研究面上项目(2012FB135); 国家自然科学基金(31160193)

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

MAO Pujia^1,2, FENG Mengdie¹, HONG Yu³, XU Zeyang¹, ZHAO Jihua³, YANG Hongwen³, SONG Wuzhan³, WANG Jiai², RAO Xiancai⁴, HUANG Fen¹, JING Shenrong¹, ZENG Weikun^1,2

1. Department of Medical Microbiology, Medical Faculty, Kunming University of Science and Technology, Kunming 650500, Yunnan, China;
2. Department of Clinical Laboratory, Medical Faculty, Kunming University, Kunming 650214, Yunnan, China;
3. Department of Nuclear Medicine, Kunming General Hospital of Chengdu Military Command, Kunming 650032, Yunnan, China;
4. Department of Microbiology, Third Military Medical University, Chongqing 404100, China

Received:2014-12-05 Revised:2015-03-23 Online:2015-07-10 Published:2015-07-10

摘要/Abstract

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pAT质粒连接, 构建pAT-副甲基因文库;扩增噬菌体早期基因gp23, 与pBT构建pBT-gp23作为诱饵质粒;将pAT-副甲基因文库和pBT-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LacZ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。

关键词: 噬菌体, 细菌双杂交, 甲型副伤寒杆菌

Abstract: Objective To screen the host protein interacting with early protein GP23 of bacteriophage LSPA1 of Salmonella Paratyphi A (S.Paratyphi A) CMCC50973. Methods The genome of S.Paratyphi A (CMCC50973) was digested by Sau3AⅠand the recovered bands (250 to 1 500 bp) were ligated with pAT plasmid to create the S.Paratyphi A genomic library. gp23, an early gene of phage LSPA1 genome, was ligated with pBT as a bait plasmid (pBT-gp23). The S.Paratyphi A genomic library plasmids and pBT-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 LacZ. 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.

Key words: Bacterial two-hybrid, S.Paratyphi A, Phage

中图分类号:

Q939.47

毛普加, 冯梦蝶, 洪愉, 许泽仰, 赵继华, 杨洪文, 宋武战, 王纪爱, 饶贤才, 黄芬, 井申荣, 曾韦锟. 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[J]. 山东大学学报（医学版）, 2015, 53(7): 29-33.

MAO Pujia, FENG Mengdie, HONG Yu, XU Zeyang, ZHAO Jihua, YANG Hongwen, SONG Wuzhan, WANG Jiai, RAO Xiancai, HUANG Fen, JING Shenrong, ZENG Weikun. Screening of host protein interacting with early protein GP23 of bacteriophage LSPA1 of Salmonella Paratyphi A[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2015, 53(7): 29-33.

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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

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