伤寒沙门菌线性质粒pBSSB1的p17基因的生物学功能分析

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 168-173.

伤寒沙门菌线性质粒pBSSB1的p17基因的生物学功能分析

朱云霞¹, 吴佳¹, 龚明玉¹, 侯书宁¹, 张绮思¹, 陈敏洁¹, 张海方^{1, 2}

1.江苏大学基础医学与医学技术学院医学生物化学系,镇江 212013; 2.苏州大学附属第二医院检验科,苏州 215004

收稿日期:2014-02-25 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:朱云霞,女,硕士研究生,研究方向：病原菌分子致病机制
基金资助:
国家自然科学基金项目（31000076）,中国博士后科学基金项目（2013M531278）,江苏省博士后科研资助计划项目（1202011B）,江苏大学大学生科研立项资助项目（12A123,13A239）

A Preliminary Study on the Biological Function of a Gene p17 Located on a Linear Plasmid pBSSB1 of Salmonella enterica serovar Typhi

Zhu Yunxia¹, Wu Jia¹, Gong Mingyu¹, Hou Shuning¹, Zhang Qisi¹, Chen Minjie¹, Zhang Haifang^{1, 2}

1. Department of Biochemistry and Molecular Biology,School of Medical Science and Laboratory Medicine,Jiangsu University,Zhenjiang 212013; 2. Department of Clinical Laboratory,the Second Affiliated Hospital of Soochow University,Suzhou 215004

Received:2014-02-25 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

摘要： H：z66阳性伤寒沙门菌含有一特有的介导鞭毛单向相变换的线性质粒pBSSB1,其上第17号基因（p17）编码一未知功能蛋白（P17）。利用λ-Red系统制备p17基因缺陷变异株,测定变异株和野生株的生长曲线,比较其生长差异,同时通过半固体LB培养基进行动力试验比较其动力差异。利用PCR技术扩增获得p17基因,构建其重组表达载体pET28a（+）-p17,通过镍柱纯化目的蛋白P17-His6,纯化后蛋白作为抗原免疫兔子以制备多克隆抗体。结果显示,制备了p17基因缺陷变异株,变异株的生长明显迟缓于野生株（P<0.05）,变异株的动力明显减弱。构建了重组表达载体pET28a（+）-p17,纯化获得了高纯度的目的蛋白P17-His6并制得相应的多克隆抗体。

关键词: 伤寒沙门菌, 线性质粒, pBSSB1, p17

Abstract: pBSSB1 is a linear plasmid which mediates the flagellar phase variation in H：z66 positive Salmonella enterica serovar Typhi（S. Typhi）. The gene named p17 is located on pBSSB1 and it encodes the protein P17 whose function is unknown. The p17 deleted mutant of S. Typhi was prepared through the λ-Red recombination system. The growth curves of wild type and p17 mutant strain were detected to compare their growth ability. The motility of wild type and p17 mutant strains was compared through the experiments on the semi-solid LB plates. The specific primers were designed to amplify the gene p17 by PCR. The amplicon was inserted into the expression vector pET28a（+）to construct recombinant vector pET28a（+）-p17, which was then transferred to E. coli BL21（DE3）to be expressed. The recombinant protein P17-His₆ was purified with Ni-TED packed column and was used as immunogen to prepare the rabbit anti-P17 polyclonal antibody. The results showed that p17 deleted mutant of S. Typhi was constructed successfully. It was showed that the growth of p17 mutant strain was significantly slower compared with the wild type strain（P<0.05）. The motility of p17 mutant strain was decreased obviously compared to the wild type strain. The gene p17 of S. Typhi was inserted into the vector pET-28a（+）and was expressed in E. coli BL21（DE3）. The rabbit anti-P17 polyclonal antibody was prepared.

Key words: Salmonella enterica serovar, Typhi Linear plasmid, pBSSB1, p17

朱云霞, 吴佳, 龚明玉, 侯书宁, 张绮思, 陈敏洁, 张海方. 伤寒沙门菌线性质粒pBSSB1的p17基因的生物学功能分析[J]. 生物技术通报, 2014, 0(10): 168-173.

Zhu Yunxia, Wu Jia, Gong Mingyu, Hou Shuning, Zhang Qisi, Chen Minjie, Zhang Haifang. A Preliminary Study on the Biological Function of a Gene p17 Located on a Linear Plasmid pBSSB1 of Salmonella enterica serovar Typhi[J]. Biotechnology Bulletin, 2014, 0(10): 168-173.

参考文献

[1] P, Wain J, Roberts M, et al. The molecular mechanisms of severe typhoid fever[J]. Trends Microbiol, 2001, 9（7）：316-320.
[2] 沙门菌黏附和侵袭肠黏膜细胞的分子基础[J]. 生命的化学, 2011, 31（1）：124-127.
[3] S, Hardy J, Sanderson KE, et al. A novel linear plasmid mediates flagellar variation in Salmonella typhi[J]. PLoS Pathog, 2007, 3（5）：e59.
[4] S, Holt K, Whitehead S, et al. A linear plasmid truncation induces unidirectional flagellar phase change in H：z66 positive Salmonella typhi[J]. Mol Microbiol, 2007, 66（5）：1207-1218.
[5] 黄新祥, 张晓磊, 等. 抗H：z66抗体诱导z66+伤寒沙门菌鞭毛相变换特点的探讨[J]. 苏州大学学报：医学版, 2010, 30（6）：1171-1175.
[6] 沙门菌鞭毛基因表达的相变换研究进展[J]. 安徽农业科学, 2010, 38（34）：19254-19255, 19265.
[7] X, Huang X, Xu S, et al. Identification of fljA located on a linear plasmid as a repressor gene of fliC in Salmonella enterica serovar Typhi[J]. Microbiol Immunol, 2009, 53（4）：191-197.
[8] S, Zou X, Sheng X et al. Expression of fljB：z66 on a novel linear plasmid of Salmonella enterica serovar Typhi is dependent on FliA and FlhDC and regulated by OmpR[J]. Braz J Microbiol, 2010, 41（3）：729-740.
[9] CM, Lutkenhaus J. Soj（ParA）DNA binding is mediated by conserved arginines and is essential for plasmid segregation[J]. Proc Natl Acad Sci USA, 1999, 96（1）：73-78.
[10] UM, Pappas KM, Winans SC. The ABCs of plasmid replication and segregation[J]. Nat Rev Microbiol, 2012, 10（11）：755-765.
[11] 张晓磊, 翁晓琴, 等.OxyR蛋白不同应激条件下伤寒沙门菌生存能力的影响[J]. 江苏大学学报：医学版. 2013, 23（2）：93-98.
[12] 高宇琳, 黄新祥, 等. 伤寒沙门菌鞭毛素基因fljB：z66的克隆表达及其多克隆抗体的制备[J]. 江苏大学学报：医学版, 2009, 19（5）：376-379.
[13] 赵昕, 张绮思, 张海方. 细菌线性质粒的复制研究 [J]. 生物技术通报, 2014（5）：32-36.
[14] T, Otake N, Yonehara H, et al. Isolation and characteri-zation of plasmids from Streptomyces[J]. J Antibiot, 1979, 32（12）：1348-1350.
[15] S, Zhang H, Sheng X, et al. Transcriptional expression of fljB：z66, a flagellin gene located on a novel linear plasmid of Salmonella enterica serovar Typhi under environmental stresses[J]. New Microbiol, 2008, 31（2）：241-247.
[16] X, Zhu Y, Zhang H, et al. Transcriptional expression of 6 genes located on pBSSB1 of Salmonella enterica serovar Typhi in different growth phases and environmental stresses[J]. Curr Microbiol, 2014, 69（3）：252-257.
[17] H, Ni B, Zhao X, et al. Fis is essential for the stability of linear plasmid pBSSB1 and affects the motility of Salmonella enterica serovar Typhi[J]. PLoS One, 2012, 7：e37462.
[18] R, Peng S, Qin Z. Two internal origins of replication in Streptomyces linear plasmid pFRL1[J]. Appl Environ Microbiol, 2010, 76（17）：5676-5683.