[1]袁橙,郭长明,左伟勇,等.制备大肠杆菌菌蜕的方法比较[J].江苏农业学报,2020,(02):410-416.[doi:doi:10.3969/j.issn.1000-4440.2020.02.022]
　YUAN Cheng,GUO Chang-ming,ZUO Wei-yong,et al.Comparison of preparation methods for of bacterial ghosts from Escherichia coli[J].,2020,(02):410-416.[doi:doi:10.3969/j.issn.1000-4440.2020.02.022]
点击复制

制备大肠杆菌菌蜕的方法比较()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年02期
页码:
410-416
栏目:
畜牧兽医·水产养殖
出版日期:
2020-04-30

文章信息/Info

Title:
Comparison of preparation methods for of bacterial ghosts from Escherichia coli
作者:
袁橙1; 郭长明1; 左伟勇1; 郝福星1; 左沁丹1; 蔺辉星2
（1.江苏农牧科技职业学院动物医学院，江苏泰州225300；2.南京农业大学动物医学院/教育部动物健康与食品安全国际合作联合实验室/农业部动物细菌学重点实验室，江苏南京210095）
Author(s):
YUAN Cheng1; GUO Chang-ming1; ZUO Wei-yong1; HAO Fu-xing1; ZUO Qin-dan1; LIN Hui-xing2
(1.College of Veterinary Medicine, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300, China;2.College of Veterinary Medicine, Nanjing Agricultural University/MOE Joint International Research Laboratory of Animal Health and Food Safety /Key Lab of Animal Bacteriology, Ministry of Agriculture, Nanjing 210095, China)
关键词:
菌蜕; 大肠杆菌; E基因; 金黄色葡萄球菌核酸酶A; 裂解效率
Keywords:
bacterial ghost; Escherichia coli; gene E; Staphylococcus aureus nuclease A; lysis efficiency
分类号:
S852.5+2
DOI:
doi:10.3969/j.issn.1000-4440.2020.02.022
文献标志码:
A
摘要:
为进一步研究大肠杆菌菌蜕的制备技术，以pBV221、pCold IV和pETDuet1载体为基础构建了表达噬菌体E基因的溶菌质粒，并利用pETDuet1的2个多克隆位点构建了同时表达E基因和金黄色葡萄球菌核酸酶A的双表达溶菌质粒。经检测，pBV221-E/DH5α组和pCold-E/BL21组的裂解效率最高，但pCold-E/BL21组的裂解起始时间晚于其他几组。诱导剂的浓度对pETDuet1-E-SNA/BL21（DE3）的裂解效率有较大影响。电泳结果显示，随着E基因的表达，细菌DNA逸出膜外，同时金黄色葡萄球菌核酸酶A将DNA降解为250 bp以下的小片段。除pBV221-E/BL21组外，β-丙内酯可实现对其他试验组菌蜕的完全灭活。电镜观察发现，几组菌蜕在结构上并无明显差别。与对照菌相比，菌蜕结构完整，电子密度低且不均匀，细胞膜有不同程度的皱缩。
Abstract:
In order to further study the preparation technology of bacterial ghosts from Escherichia coli, the bacteriolytic plasmids expressing bacteriophage E gene were constructed based on vectors including pBV221, pCold IV and pETDuet1. Using two polychonal sites of pETDuet1, a double expression plasmid expressing E gene and Staphylococcus aureus nuclease A was constructed. The lysis efficiency of pBV221-E/DH5α group and pCold-E/BL21 group was the highest, but the lysis initiation time of pCold-E/BL21 group was later than other groups. The concentration of inducer had a great influence on the lysis efficiency of pETDuet1-E-SNA/BL21(DE3). Electrophoresis results showed that with the expression of E gene, bacterial DNA escaped from the cell membrane. Meanwhile, Staphylococcus aureus nuclease A degraded DNA to a small fragment below 250 bp. The β-propiolactone could completely inactivate the bacterial ghost of experimental groups expect pBV221-E/BL21 group. Electron microscopic observation indicated that there was no significant difference in the structure of the bacterial ghost among different groups. Compared with Escherichia coli, the electron density of bacterial ghost was lower and uneven, the cell membrane shrank, and the structure was complete.

参考文献/References:

[1]LUBITZ W, PUGSLEY A P. Changes in host cell phospholipid composition of X174 gene E product[J]. Fems Microbiology Letters, 1985, 30(1/2):171-175.
[2]彭凌,杨旭夫.重组双基因鸭源大肠杆菌菌蜕的制备[J].中国人兽共患病学报,2018,34(7):639-642.
[3]SZOSTAK M P, HENSEL A, EKO F O, et al. Bacterial ghosts: non-living candidate vaccines[J]. Journal of Biotechnology, 1996, 44(1-3):161-170.
[4]HENSEL A, HUTER V, KATINGER A, et al. Intramuscular immunization with genetically inactivated (ghosts) Actinobacillus pleuropneumoniae serotype 9 protects pigs against homologous aerosol challenge and prevents carrier state[J]. Vaccine, 2000, 18(26):2945-2955.
[5]MAYR U B, HALLER C, HAIDINGER W, et al. Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157∶H7 bacterial ghosts protects mice against lethal challenge[J]. Infection & Immunity, 2005, 73(8):4810.
[6]MARCHART J, DROPMANN G, LECHLEITNER S, et al. Pasteurella multocida - and Pasteurella haemolytica -ghosts: new vaccine candidates[J]. Vaccine, 2003, 21(25):3988-3997.
[7]SERYUN K, SUNGKOO Y K, KIHONG K. Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts.[J]. Fish & Shellfish Immunology, 2006, 20(4):621-626.
[8]胡剑刚,董洪亮,付立霞,等.制备禽致病性大肠杆菌菌蜕的三种方法比较[J].生物工程学报,2017,33(12):2009-2016.
[9]孙颖. App菌影装载多杀性巴氏杆菌OmpH外膜蛋白DNA疫苗的研究[D]. 天津: 天津农学院,2011.
[10]萨姆布鲁克J, 格林M R. 分子克隆实验指南[M].4版.北京: 科学出版社, 2017: 138-142.
[11]WENXING Z, YUYU Z, XINLI L. Efficient production of safety-enhanced Escherichia coli ghosts bytandem expression of PhiX 174 mutant gene E and staphylococcalnuclease A gene[J]. Microbiological Research, 2015, 176：7-13.
[12]曹春光. 苏鲁部分地区禽致病性大肠杆菌的分离及2个O78分离株致病性的研究[D].扬州: 扬州大学,2015.
[13]VILTE D A, LARZBAL M, MAYR U B, et al. A systemic vaccine based on Escherichia coli O157∶H7 bacterial ghosts (BGs) reduces the excretion of E. coli O157∶H7 in calves[J]. Veterinary Immunology & Immunopathology, 2012, 146(2):169-176.
[14]LAGZIAN M, BASSAMI M R, DEHGHANI H. In vitro responses of chicken macrophage-like monocytes following exposure to pathogenic and non-pathogenic E. coli ghosts loaded with a rational design of conserved genetic materials of influenza and Newcastle disease viruses[J]. Veterinary Immunology & Immunopathology, 2016, 176:5-17.
[15]TUNTUFYE H N, ONS E, PHAM A D, et al. Escherichia coli ghosts or live E. coli expressing the ferri-siderophore receptors FepA, FhuE, IroN and IutA do not protect broiler chickens against avian pathogenic E. coli (APEC)[J]. Veterinary Microbiology, 2012, 159(3/4):470-478.
[16]张文文,王小敏,肖琦,等. 猪TTV2 ORF1基因的原核表达及多克隆抗体的制备[J].江苏农业学报,2014,30(1):119-124.
[17]李小妹,马跃,李安兴,等. 禽致病性大肠杆菌(APEC)菌蜕的制备研究[J].中国预防兽医学报,2007,20(6):423-427.
[18]徐守振,尹燕博,王新. 动物疫苗中常用抗原灭活剂的研究进展[J].中国畜牧兽医,2010,37(9):162-167.
[19]付立霞,张磊,李欣容,等. 金黄色葡萄球菌核酸酶A在温控双表达载体中的表达及其活性分析[J].生物学杂志,2018,35(2):34-37.
[20]李鹏成,孙冰,乔绪稳,等. 乳酸乳球菌肽聚糖锚钩蛋白原核表达载体的构建与表达[J].江苏农业学报,2013,29(6):1399-1404.
[21]吕敏娜,覃宗华,余劲术,等. 鸭源大肠杆菌O_(78)菌蜕的制备及免疫原性研究[J].中国预防兽医学报,2010,32(9):712-715.

相似文献/References:

[1]王翔宇,宋玉慧,荆炜,等.鸡源大肠杆菌 BlaCTX鄄M鄄125型 ESBL 基因的定位及遗传环境[J].江苏农业学报,2017,(01):141.[doi:10.3969/j.issn.1000-4440.2017.01.022 ]
　WANG Xiang-yu,SONG Yu-hui,JING Wei,et al.The location and genetic environment of blaCTX-M-125 ESBL gene from Escherichia coli in chickens[J].,2017,(02):141.[doi:10.3969/j.issn.1000-4440.2017.01.022 ]
[2]陆玉建,张韩杰,刘南南.大肠杆菌 otsA 基因的克隆和转化本生烟草[J].江苏农业学报,2015,(01):32.[doi:10.3969/j.issn.1000-4440.2015.01.005]
　LU Yu-jian,ZHANG Han-jie,LIU Nan-nan.Cloning of otsA gene in Escherichia coli and its transformation into Nicotiana benthamiana[J].,2015,(02):32.[doi:10.3969/j.issn.1000-4440.2015.01.005]
[3]钟希娜,何涛,葛展霞,等.一种对mcr-1阳性大肠杆菌具有裂解作用的噬菌体生物学特性[J].江苏农业学报,2018,(02):452.[doi:doi:10.3969/j.issn.1000-4440.2018.02.033]
　ZHONG Xi-na,HE Tao,GE Zhan-xia,et al.Characterization of a lytic bacteriophage infecting mcr-1-positive Escherichia coli[J].,2018,(02):452.[doi:doi:10.3969/j.issn.1000-4440.2018.02.033]
[4]李陇平,杨吉,白蹉蹉,等.一株绒山羊源大肠杆菌噬菌体φPTK的分离鉴定及生物学特性分析[J].江苏农业学报,2018,(04):847.[doi:doi:10.3969/j.issn.1000-4440.2018.04.019]
　LI Long-ping,YANG Ji,BAI Cuo-cuo,et al.Isolation, identification and biological properties of a lytic phage against goat (Capra hircus)-associated Escherichia coli[J].,2018,(02):847.[doi:doi:10.3969/j.issn.1000-4440.2018.04.019]
[5]王警,张雪寒,郭芸芸,等.江苏地区奶牛场blaNDM阳性大肠杆菌的分子流行病学[J].江苏农业学报,2020,(02):391.[doi:doi:10.3969/j.issn.1000-4440.2020.02.019]
　WANG Jing,ZHANG Xue-han,GUO Yun-yun,et al.Molecular epidemiological study on blaNDM-positive Escherichia coli in dairy farms in Jiangsu province[J].,2020,(02):391.[doi:doi:10.3969/j.issn.1000-4440.2020.02.019]

备注/Memo

备注/Memo:
收稿日期：2019-08-24基金项目：江苏省高等学校自然科学研究面上项目（17KJB230003）；江苏省高校优秀科技创新团队项目（201753）；江苏农牧科技职业学院科研项目（NSF201605）作者简介：袁橙(1986-)，女，江苏盐城人，博士，讲师，从事细菌菌蜕制备和寄生虫分子免疫研究。(E-mail)840289909@qq.com通讯作者：蔺辉星，(E-mail)linhuix@ 163. com
更新日期/Last Update: 2020-05-18