抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性

doi:10.13481/j.1671-587x.20150213

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (02): 275-281.doi: 10.13481/j.1671-587x.20150213

抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性

毛普加^1,2, 冯梦蝶¹, 洪愉³, 毛小萍⁴, 许泽仰¹, 赵继华³, 杨洪文³, 宋武战³, 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 650032;
4. 湖北省孝感市中心医院泌尿外科, 湖北孝感 432000

收稿日期:2014-08-11 出版日期:2015-03-28 发布日期:2015-04-04
通讯作者: 曾韦锟, 讲师, 硕士研究生导师(Tel:0871-65920776, E-mail:zengweikun@gmail.com) E-mail:zengweikun@gmail.com
作者简介:毛普加(1989-), 男, 湖北省武穴市人, 在读理学硕士, 主要从事病原微生物方面的研究。
基金资助:
国家自然科学基金资助课题(31160193);云南省教育厅科研项目资助课题(2010Y398);云南省科技厅应用基础研究面上项目资助课题(2010ZC055;2012FB135)

Construction of anti-phage Salmonella Paratyphi A mutant strains and its biological characteristics

MAO Pujia^1,2, FENG Mengdie¹, HONG Yu³, MAO Xiaoping⁴, XU Zeyang¹, ZHAO Jihua³, YANG Hongwen³, SONG Wuzhan³, HUANG Fen¹, JING Shenrong¹, ZENG Weikun^1,2

1. Department of Medical Microbiology, School of Medical Science, Kunming University of Science and Technology, Kunming 650500, China;
2. Department of Clinical Laboratory Science, Medical Faculty, Kunming University, Kunming 650214, China;
3. Department of Nuclear Medicine, Kunming General Hospital, Chengdu Military Command, Kunming 650032, China;
4. Department of Urinary Surgery, Xiaogan General Hospital, Hubei Province, Xiaogan 432000, China

Received:2014-08-11 Online:2015-03-28 Published:2015-04-04

摘要/Abstract

摘要：

目的:采用同源重组基因敲除方法构建3株甲型副伤寒沙门菌(副甲)抗噬菌体突变菌,并研究其生物学特性。方法:通过PCR和融合PCR方法获得了2、3、5号同源臂片段,经酶切后与pYG4载体连接,构建pYG4-2、pYG4-3和pYG4-5同源重组质粒。经PCR测序鉴定正确后,与野生型副甲菌进行同源重组,获得2、3、5号3株抗噬菌体突变菌。将3株突变菌和野生型副甲菌经不同种类和浓度的抗生素、不同pH值和温度及不同浓度的盐离子处理后,测量菌株的A₅₉₅值,分析3株突变菌对抗生素、温度、pH值和盐离子的敏感性以及生长曲线。结果:PCR和融合PCR扩增出3个同源臂,构建的pYG4-2、pYG4-3和pYG4-5同源重组质粒经PCR测序表明构建成功;同源重组和抗噬菌体验证表明成功获得3株抗噬菌体突变菌。3株突变菌产生了卡那霉素抗性,且对链霉素表现出敏感性;2号菌表现出氨苄青霉素抗性,5号菌表现高温度耐受性;与野性型副甲菌比较,3株突变菌的生长曲线明显平缓,尤以2号突变菌最为突出。结论:成功构建抗噬菌体突变菌,并获得一些特殊的生物学特性。

关键词: 甲型副伤寒沙门菌, 突变菌, 同源重组, 生物学特性, 噬菌体抗性

Abstract:

Objective To construct three strains of anti-phage Salmonella Paratyphi A mutants by homologous recombination and knocking out genes in wild Salmonella Paratyphi A, and to analyze their biological characteristics.Methods The No.2,3,5 homology arm fragments obtained by PCR and fusion PCR were connected with pYG4 vector after digestion to construct the pYG4-2,pYG4-3,and pYG4-5 homologous recombination plasmids. Three strains of anti-phage mutants (No.2,3,5) were obtained after the plasmids were identificated by PCR sequencing and recombinated with wild Salmonella Paratyphi A. After the three mutant strains and wild Salmonella Paratyphi A were treated with different kinds and concentrations of antibiotics,different pH values and temperature and different concentrations of salt ions,respectively;the A₅₉₅ values were measured,and their sensitivities to the antibiotics,temperature,pH values and salt ions and the growth curves were analyzed.Results The results of PCR and fusion PCR amplification suggested that three homology arms were obtained. The PCR sequencing showed that the homologous recombinant plasmids pYG4-2,pYG4-3,and pYG4-5 were constructed successfully.The homologous recombination and anti-phage verification implied that three strains of anti-phage mutants were successfully gained. Three strains of anti-phage mutants gained kanamycin resistance and showed highly sensitivity to streptomycin;the No.2 mutant strain showed ampicillin resistance and the No.5 mutant strain performanced a high temperature tolerance. In addition,the growth curves of three mutants became obviously placid than that of wild Salmonella Paratyphi A,especially in the No.2 mutant.Conclusion The anti-phage mutants are successfully constructed and get some specially biological chatacteristics.

Key words: biological characteristics, phage resistance, Salmonella Paratyphi A, mutant,homologous recombination

中图分类号:

R378.22

毛普加, 冯梦蝶, 洪愉, 毛小萍, 许泽仰, 赵继华, 杨洪文, 宋武战, 黄芬, 井申荣, 曾韦锟. 抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性[J]. 吉林大学学报(医学版), 2015, 41(02): 275-281.

MAO Pujia, FENG Mengdie, HONG Yu, MAO Xiaoping, XU Zeyang, ZHAO Jihua, YANG Hongwen, SONG Wuzhan, HUANG Fen, JING Shenrong, ZENG Weikun. Construction of anti-phage Salmonella Paratyphi A mutant strains and its biological characteristics[J]. Journal of Jilin University Medicine Edition, 2015, 41(02): 275-281.

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抗噬菌体甲型副伤寒沙门菌突变菌的构建及其生物学特性

Construction of anti-phage Salmonella Paratyphi A mutant strains and its biological characteristics

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