鼠伤寒沙门菌LH430株asd基因缺失株平衡致死系统的构建及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2019.11.024

中国畜牧兽医 ›› 2019, Vol. 46 ›› Issue (11): 3341-3349.doi: 10.16431/j.cnki.1671-7236.2019.11.024

鼠伤寒沙门菌LH430株asd基因缺失株平衡致死系统的构建及生物学特性分析

张明亮^1,2,3, 孙长江², 顾敬敏², 崔子寅², 韩文瑜²

1. 安阳工学院生物与食品工程学院, 安阳 455000;
2. 吉林大学动物医学学院, 长春 130000;
3. 河南省动物疫病防控与营养免疫院士工作站, 安阳 455000

收稿日期:2019-04-04 出版日期:2019-11-20 发布日期:2019-11-25
通讯作者: 韩文瑜 E-mail:hanwy@jlu.edu.cn
作者简介:张明亮(1985-),男,河南遂平人,博士,研究方向:病原微生物和分子免疫学,E-mail:mingliang90909@163.com;孙长江(1978-),男,吉林梅河口人,博士,研究方向:人畜共患传染病防治,E-mail:18021346@qq.com
基金资助:
国家高技术研究发展计划项目（2011AA10A210）；国家自然科学基金青年基金项目（31802170）；安阳工学院博士科研启动基金项目（BSJ2016013）

Construction and Biological Characteristics Analysis of asd Gene Mutant Balanced Lethal Host-vector System of Salmonella Typhimurium LH430 Strain

ZHANG Mingliang^1,2,3, SUN Changjiang², GU Jingmin², CUI Ziyin², HAN Wenyu²

1. College of Biological Science and Food Engineering, Anyang Institute of Technology, Anyang 455000, China;
2. College of Veterinary Medicine, Jilin University, Changchun 130000, China;
3. Academician Workstation of Animal Disease Control and Nutrition Immunity in Henan Province, Anyang 455000, China

Received:2019-04-04 Online:2019-11-20 Published:2019-11-25

摘要/Abstract

摘要： 本研究旨在构建基于减毒鼠伤寒沙门菌的平衡致死系统。将打靶基因片段Cat基因重组入鼠伤寒沙门菌LH430株asd基因位置，之后将温敏型质粒pCP20转入菌体中用以消除Cat基因片段，通过PCR技术两步法筛选出缺失株Δasd LH430；将asd⁺的原核表达质粒pYA3493转入Δasd LH430，经PCR及测序鉴定表明鼠伤寒沙门菌平衡致死系统Δasd LH430（pYA3493）构建成功。对构建的基因缺失株研究表明，该缺失株失去了在二氨基庚二酸（DAP）阴性环境中生存的能力；糖发酵试验表明，缺失菌株在利用碳源的能力方面与亲本菌株保持一致；9种生化试验结果显示，缺失菌株遗传了亲本菌株的生化特性；遗传稳定性试验表明，缺失菌株能够稳定遗传缺失的423 bp的基因片段；将绿色荧光基因转入所构建的平衡致死系统，成功构建携带绿色荧光基因的重组菌Δasd LH430（pYA-gfp），对其研究表明该重组菌能够有效表达绿色荧光蛋白。本研究成功构建了鼠伤寒沙门菌平衡致死系统Δasd LH430（pYA3493），能够有效表达所携带的基因，该系统可作为疫苗活载体来携带外源基因。

关键词: 鼠伤寒沙门菌; 平衡致死系统; 活疫苗载体; 生物学特性

Abstract: This study was aimed to construct a balanced lethal system based on attenuated Salmonella Typhimurium.Cat gene was recombined into the position of asd gene in Salmonella Typhimurium LH430 strain,then the thermo sensitive plasmid pCP20 was transferred into the strain to eliminate the Cat gene fragment,and the mutant strain Δasd LH430 was identified by PCR.Then the balanced lethal system of Salmonella Typhimurium Δasd LH430(pYA3493) was successfully constructed with the plasmid pYA3493 transferred into Δasd LH430.The mutant strain lost the ability to survive in the medium without DAP,had the same ability of using carbon sources was consistent with parent strains,and inherited the biochemical characteristics from parent strains based on nine biochemical experiments,Δasd LH430(pYA-gfp) carrying green fluorescent gene was successfully constructed,and the green fluorescent protein could be also inherited the deletion fragment of 423 bp.Then the Δasd LH430(pYA-gfp) carrying green fluorescent gene was successfully constructed,and the green fluorescent protein could be effectively expressed by Δasd LH430(pYA-gfp).In conclusion,the balanced lethal system Δasd LH430(pYA3493) was successfully constructed in this study,and could effectively express the carrying genes,the system could be used as a living vaccine vector to carry exogenous genes.

Key words: Salmonella Typhimurium; balanced lethal system; living vaccine vector; biological characteristics

中图分类号:

R378.2⁺3

张明亮, 孙长江, 顾敬敏, 崔子寅, 韩文瑜. 鼠伤寒沙门菌LH430株asd基因缺失株平衡致死系统的构建及生物学特性分析[J]. 中国畜牧兽医, 2019, 46(11): 3341-3349.

ZHANG Mingliang, SUN Changjiang, GU Jingmin, CUI Ziyin, HAN Wenyu. Construction and Biological Characteristics Analysis of asd Gene Mutant Balanced Lethal Host-vector System of Salmonella Typhimurium LH430 Strain[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(11): 3341-3349.

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鼠伤寒沙门菌LH430株asd基因缺失株平衡致死系统的构建及生物学特性分析

Construction and Biological Characteristics Analysis of asd Gene Mutant Balanced Lethal Host-vector System of Salmonella Typhimurium LH430 Strain

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