SG97A平衡致死系统的构建及其初步应用

doi:10.11843/j.issn.0366-6964.2015.12.016

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (12): 2243-2250.doi: 10.11843/j.issn.0366-6964.2015.12.016

SG97A平衡致死系统的构建及其初步应用

耿士忠^#，陈晓娟^#，潘志明，焦新安^*

（扬州大学江苏省人兽共患病学重点实验室/江苏省动物重要疫病与人兽共患病防控协同创新中心，扬州 225009）

收稿日期:2015-04-13 出版日期:2015-12-23 发布日期:2015-12-18
通讯作者: 焦新安，教授，E-mail：jiao@yzu.edu.cn
作者简介:耿士忠(1972-)，男，江苏高邮人，副教授，博士，主要从事沙门菌致病机制研究及疫苗开发，Tel：0514-87971136，E-mail：gszzsg115@163.com。耿士忠、陈晓娟对本文贡献相同
基金资助:
公益性行业(农业)项目(201403054)；“863”项目(2011AA10A212)；中国博士后基金（2014M551670）；江苏省重点实验室开放课题(0273896034819)；江苏省博士后基金(1302067C)；扬州大学创新培育基金(2013CXJ070)

Construction and Preliminary Application of Balanced Lethal System Based on Attenuated Salmonella SG97A

GENG Shi-zhong^#，CHEN Xiao-juan^#，PAN Zhi-ming，JIAO Xin-an^*

(Jiangsu Key Laboratory of Zoonoses，Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses，Yangzhou University，Yangzhou 225009，China)

Received:2015-04-13 Online:2015-12-23 Published:2015-12-18

摘要/Abstract

摘要：

敲除鸡伤寒沙门菌(SG)减毒疫苗株97A asd基因，构建宿主载体平衡致死系统并表达新城疫病毒(NDV)F蛋白，构建二价活菌苗。PCR扩增两段位于asd基因外侧的序列作为等位基因，以氯霉素抗性(Cm^R)基因替代asd基因，构建自杀质粒pGMB151Δasd：：Cm^R，通过E.coli χ7213与SG97A固相杂交，将质粒转入SG97A，反向筛选获得SG97AΔasd：：Cm^R，利用质粒pCP20去除Cm^R后，DAP的依赖菌株命名为SG97AΔasd。将含asd基因的质粒pYA3334转入SG97AΔasd中构建宿主载体平衡致死系统，并表达NDV-F蛋白。结果表明，通过等位基因重组方法，成功构建SG97AΔasd及宿主载体平衡致死系统，重组菌能够表达NDV-F蛋白，并能在鸡体内诱导产生特异性抗体。本研究成功构建SG97AΔasd宿主载体平衡致死系统，并表达NDV-F蛋白及其诱导宿主产生抗体，为应用该系统构建二价活菌苗的研究奠定了基础。

关键词: 鸡伤寒沙门菌, asd基因, 宿主载体平衡致死系统, NDV-F蛋白

Abstract:

The aim of this study was to delete asd gene of Salmonella gallinarum (SG) attenuated vaccine strain 97A and construct bivalent live vaccine based on host-vector balanced lethal system to express F protein of Newcastle disease virus(NDV).Two DNA fragments flanking asd gene as allele were amplificated by PCR and asd gene was replaced by chloramphenicol resistance(Cm^R) gene to construct the suicide plasmid pGMB151Δasd：：Cm^R.After conjugation，the recombiant plasmid was transformed into SG97A from E.coli χ7213，SG97AΔasd：：Cm^R was reversely screened out under the selectable pressure of chloramphenicol，DAP-dependent strain was named SG97AΔasd.Vector-host balanced lethal system was construct by pYA3334 containing asd gene being transferred into SG97AΔasd and to expressed F protein of NDV.SG97AΔasd was successfully constructed by allelic exchanges，its growth was DAP-dependent.Vector-host balanced lethal system was constructed by pYA3334 being transferred to SG97AΔasd and could express F protein of NDV and also induce specific antibodies in infected chickens.Vector-host balanced lethal system was constructed successfully based on attenuated Salmonella SG97AΔasd in this study and could express F gene of NDV as foreign gene to induce special antibodies in animal model，it laid a foundation for bivalent live vaccine based on attenuated Salmonella.

Key words: Salmonella gallinarum(SG), asd gene, vector-host balanced lethal system, NDV-F protein

中图分类号:

S852.612

耿士忠，陈晓娟，潘志明，焦新安. SG97A平衡致死系统的构建及其初步应用[J]. 畜牧兽医学报, 2015, 46(12): 2243-2250.

GENG Shi-zhong，CHEN Xiao-juan，PAN Zhi-ming，JIAO Xin-an. Construction and Preliminary Application of Balanced Lethal System Based on Attenuated Salmonella SG97A[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(12): 2243-2250.

参考文献

［1］刘海侠，王宝安，潘志明，等.减毒鸡沙门氏菌疫苗株97A安全性评价［J］.中国兽医杂志，2007，43(11)：72-73.
LIU H X，WANG B A，PAN Z M，et al.Evaluation on safety to Chicken of attenuated Salmonella vaccine strain 97A［J］.Chinese Journal of Veterinary Medicine，2007，43(11)：72-73.(in Chinese)
［2］陈松彪，李静，尚珂，等.鸡白痢沙门菌C79-13 ΔcrpΔasd平衡致死系统的构建及其生物学特性的研究［J］.中国免疫学杂志，2014，30(8)：1083-1087，1092.
CHEN S B，LI J，SHANG K，et al.Construction of Salmonella Pullorum C79-13 ΔcrpΔasd mutant balanced-lethal host-vector system and its biological features［J］.Chinese Journal of Immunology，2014，30(8)：1083-1087，1092.(in Chinese)
［3］徐引弟，郭爱珍，刘维红，等.猪霍乱沙门菌C500 株ΔcrpΔasd 缺失株平衡致死载体系统的构建及鉴定［J］.生物工程学报，2006，22(3)：366-372.
XU Y D，GUO A Z，LIU W H，et al.Construction and characterization of ΔcrpΔasd Mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain［J］.Chinese Journal of Biotechnology，2006，22(3)：366-372.(in Chinese)
［4］GENG S Z，JIAO X A，PAN Z M，et al.An improved method to knock out the asd gene of Salmonella enterica serovar Pullorum［J］.J Biomed Biotechnol，2009，2009：646380.doi：10.1155/2009/646380.
［5］耿士忠，刘男男，焦新安，等.鸡白痢沙门菌S06004ΔspiC突变株的构建与鉴定［J］.中国兽医科学，2014，44(4)：379-386.
GENG S Z，LIU N N，JIAO X A.et al.Development and identification of spiC gene-deleted Salmonella pullorum mutant strain ΔspiC［J］.Chinese Veterinary Science，2014，44(4)：379-386.(in Chinese)
［6］WANG S F，KONG Q K，CURTISS R III.New technologies in developing recombinant attenuated Salmonella vaccine vectors［J］.Microb Pathog，2013，58：17-28.
［7］GALEN J E，GOMEZ-DUARTE O G，LOSONSKY G A，et al.A murine model of intranasal immunization to assess the immunogenicity of attenuated Salmonella typhi live vector vaccines in stimulating serum antibody responses to expressed foreign antigens［J］.Vaccine，1997，15(6-7)：700-708.
［8］VECINO W H，MORIN P M，AGHA R，et al.Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T cells against HIV［J］.Immunol Lett，2002，82(3)：197-204.
［9］KARPENKO L I，NEKRASOVA N A，IGNAT’EV G M，et al.Immune response in oral and rectal immunization by the attenuated strain of Salmonella carrying the HIV DNA-vaccine［J］.Vopr Virusol，2003，48(4)：16-20.
［10］PASETTI M F，LEVINE M M，SZTEIN M B，et al.Animal models paving the way for clinical trials of attenuated Salmonella enterica serovar typhi live oral vaccines and live vectors［J］.Vaccine，2003，21(5-6)：401-418.
［11］CURTISS R Ⅲ，ZHANG X，WANDA S Y，et al.Induction of host immune responses using Salmonella-vectored vaccines//Brogden K A，Minion F C，Cornick N(ed).Virulence mechanisms of bacterial pathogens［M］.4th ed.ASM Press，Washington，DC.2007：297-313.
［12］刘婷，焦新安，潘志明，等.表达破伤风毒素C片段的重组鼠伤寒沙门菌的构建与鉴定［J］.中国兽医科技，2005，35(11)：883-887.
LIU T，JIAO X A，PAN Z M，et al.Construction and identification of recombinant Salmonella typhimurium strain expressing tetanus toxin fragment C［J］.Chinese Journal of Veterinary Science and Technology，2005，35(11)：883-887.(in Chinese)
［13］SCHRIEFER A，MALTEZ J R，SILVA N，et al.Expression of a pilin subunit BfpA of the bundle-forming pilus of enteropathogenic Escherichia coli in an aroA live Salmonella vaccine strain［J］.Vaccine，1999，17(7-8)：770-778.
［14］QU D，WANG S，CAI W，et al.Protective effect of a DNA vaccine delivered in attenuated Salmonella typhimurium against Toxoplasma gondii infection in mice［J］.Vaccine，2008，26(35)：4541-4548.
［15］WANG J，MICHEL V，LECLERC C，et al.Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium ［J］.Vaccine，1999，17(1)：1-12.
［16］HOPKINS S，KRAEHENBUHL J P，SCHÖDEL F，et al.A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization［J］. Infect Immun，1995，63(9)：3279-3286.
［17］BACHTIAR E W，SHENG K C，FIFIS T，et al.Delivery of a heterologous antigen by a registered Salmonella vaccine(STM1)［J］.FEMS Microbiol Lett，2003，227(2)：211-217.
［18］徐引弟.猪霍乱沙门氏菌C500株crp^-、asd^-缺失株平衡致死系统的构建及应用［D］.武汉：华中农业大学，2006.
XU Y D.Construction and application of crp^-， asd^- deleted mutants host-vector balanced lethal system of Salmonella choleraesuis C500 strain［D］.Wuhan：Huazhong Agricultural University，2006.(in Chinese)
［19］闫一剑.迟钝爱德华氏菌平衡致死系统的构建及其在新型疫苗开发中的应用［D］.上海：华东理工大学，2013.
YAN Y J.Construction of an asd balanced-lethal system in Edwardsiella tarda and its potential application in multivalent vaccine development［D］.Shanghai：East China University of Science and Technology，2013.(in Chinese)

[1]	耿士忠，陈晓娟，潘志明，焦新安. SG97A平衡致死系统的构建及其初步应用[J]. 畜牧兽医学报, 2015, 46(12): 2243-2250.
[2]	杨磊，赵广英，窦文超. 新型凝集技术快速检测鸡白痢、鸡伤寒沙门菌[J]. 畜牧兽医学报, 2013, 44(12): 1994-1999.
[3]	杨磊，赵广英，窦文超. 新型凝集技术快速检测鸡白痢、鸡伤寒沙门菌[J]. 畜牧兽医学报, 2013, 44(12): 1994-1999.
[4]	胡春梅，窦文超，赵广英. 双重放大酶免疫传感器检测鸡白痢鸡伤寒沙门菌[J]. 畜牧兽医学报, 2012, 43(12): 1991-1997.
[5]	胡春梅，窦文超，赵广英. 双重放大酶免疫传感器检测鸡白痢鸡伤寒沙门菌[J]. 畜牧兽医学报, 2012, 43(12): 1991-1997.

SG97A平衡致死系统的构建及其初步应用

Construction and Preliminary Application of Balanced Lethal System Based on Attenuated Salmonella SG97A

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 5

编辑推荐

Metrics

本文评价