鸡白痢沙门菌pagC缺失株的构建及菌蜕制备
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摘要
为了开发具有鉴别诊断功能的鸡白痢沙门菌疫苗,通过λ-Red同源重组敲除鸡白痢沙门菌CVCC1800的pagC基因,成功获得ΔpagC菌株;合成细胞穿透肽(cell penetrating peptide,CPP)制备菌蜕并对处理条件进行了优化。结果显示:作用浓度为50μmol/L,ΔpagC菌液OD_(600 nm)=0.4左右时CPP灭活效果最好。本研究制备的基于ΔpagC鸡白痢沙门菌菌蜕,在保留抗原性的同时为鉴别诊断提供了靶点,为后续开发针对鸡白痢沙门菌的鉴别诊断菌蜕疫苗奠定了基础。
In order to develop a Salmonella pullorum vaccine with a function of differential diagnosis, the pagC gene of Salmonella pullorum CVCC1800 was knocked off by λ-Red homologous recombination, and the ΔpagC strain was successfully obtained. Synthetic cell penetrating peptides(cell penetrating peptide, CPP) was used for the preparation of bacterial ghost and the processing conditions were optimized. The results showed that the CPP was effectively inactivated when the concentration was 50 μmol/L and the ΔpagC solution was OD_(600 nm)=0.4. The ΔpagC-based Salmonella pullorum bacteria ghost prepared in this study provided a target for differential diagnosis as well as retaining antigenicity, which laid a foundation for the future development of a differential diagnosis ghost vaccine against Salmonella pullorum.
In order to develop a Salmonella pullorum vaccine with a function of differential diagnosis, the pagC gene of Salmonella pullorum CVCC1800 was knocked off by λ-Red homologous recombination, and the ΔpagC strain was successfully obtained. Synthetic cell penetrating peptides(cell penetrating peptide, CPP) was used for the preparation of bacterial ghost and the processing conditions were optimized. The results showed that the CPP was effectively inactivated when the concentration was 50 μmol/L and the ΔpagC solution was OD_(600 nm)=0.4. The ΔpagC-based Salmonella pullorum bacteria ghost prepared in this study provided a target for differential diagnosis as well as retaining antigenicity, which laid a foundation for the future development of a differential diagnosis ghost vaccine against Salmonella pullorum.
引文
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[17] Peng W,Si W,Yin L,et al.Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks[J].Immunobiology,2011,216(5):558-565.
[18] Zhao Y M,Jiang X,Qu Y Y,et al.Salmonella detection in powdered dairy products using a novel molecular tool[J].J Dairy Sci,2017,100(5):3480-3496.
[19] Alix E,Miki T,Felix C,et al.Interplay between MgtC and PagC in Salmonella enterica serovar Typhimurium[J].Microb Pathogene-sis,2008,45(3):236-240.
[20] 王宏卫,赵平,张珉,等.鼠伤寒沙门菌pagC启动子的克隆与活性分析[J].微生物学报,2003,43(3):308-314.
[21] 张佳,范欣丽,葛玉梅,等.甲型副伤寒杆菌pagC基因分布及其重组表达产物免疫保护作用[J].浙江大学学报(医学版),2013,42(2):171-176,231.
[22] 智海东,王云峰,陈洪岩.我国兽用基因工程疫苗研发现状与策略[J].动物医学进展,2011,32(6):174-178.
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[24] Murphy K C.Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J].J Bacteriol,1998,180(8):2063-2071.
[25] Murphy C N,Fowler R C,Williams A J,et al.Nontyphoidal Salmonella enterica nonsusceptible to both levofloxacin and ceftriaxone in Nebraska,United States 2014-2015[J].Foodborne Pathog Dis,2018,15(4):235-238.
[26] Murphy K C,Campellone K G,Poteete A R.PCR-mediated gene replacement in Escherichia coli[J].Gene,2000,246(1/2):321-330.
[27] Muyrers J P,Zhang Y,Testa G,et al.Rapid modification of bacterial artificial chromosomes by ET-recombination[J].Nucleic Acids Res,1999,27(6):1555-1557.
[28] 王恒,冯尔玲,史兆兴,等.用Red系统快速敲除痢疾杆菌asd基因[J].军事医学科学院院刊,2002(3):172-175.
[29] Wang X,Lu C.Mice orally vaccinate d with Edwardsiella tarda ghosts are significantly protected against infection[J].Vaccine,2009,27(10):1571-1578.
[2] Barrow P A,Freitas Neto O C.Pullorum disease and fowl typhoid-new thoughts on old diseases:a review[J].Avian Pathol,2011,40(1):1-13.
[3] Wigley P,Hulme S D,Powers C,et al.Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity[J].Infect Immun,2005,73(5):2986-2990.
[4] Jones M A,Wigley P,Page K L,et al.Salmonella enterica serovar Gallinarum requires the Salmonella pathogenicity island 2 type III secretion system but not the Salmonella pathogenicity island 1 type III secretion system for virulence in chickens[J].Infect Immun,2001,69(9):5471-5476.
[5] Kang M S,Kwon Y K,Kim H R,et al.Differential identification of Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum and the biovar Gallinarum live vaccine strain 9R[J].Vet Microbiol,2012,160(3/4):491-495.
[6] Pan Z M,Wang X Q,Zhang X M,et al.Changes in antimicrobial resistance among Salmonella enterica subspecies enterica serovar Pullorum isolates in China from 1962 to 2007[J].Vet Microbiol,2009,136(3/4):387-392.
[7] Babu U,Scott M,Myers M J,et al.Effects of live attenuated and killed Salmonella vaccine on T-lymphocyte mediated immunity in laying hens[J].Vet Immunol,2003,91(1):39-44.
[8] Liu H F,Chen L P,Wang X M,et al.Decrease of colonization in the chicks' cecum and internal organs of Salmonella enterica serovar Pullorum by deletion of cpdB by Red system[J].Microbi Pathogenesis,2015,80:21-26.
[9] Okamura M,Tachizaki H,Kubo T,et al.Comparative evaluation of a bivalent killed Salmonella vaccine to prevent egg contamination with Salmonella enterica serovars Enteritidis,Typhimurium,and Gallinarum biovar Pullorum,using 4 different challenge models[J].Vaccine,2007,25(25):4837-4844.
[10] Jalava K,Hensel A,Szostak M,et al.Bacterial ghosts as vaccine candidates for veterinary applications[J].J Control Release,2002,85(1/2/3):17-25.
[11] Muhammad A,Champeimont J,Mayr U B,et al.Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications[J].Expert Rev Vaccines,2012,11(1):97-116.
[12] Witte A,Blasi U,Halfmann G,et al.Phix174 Protein E-Mediated Lysis of Escherichia-Coli[J].Biochimie,1990,72(2/3):191-200.
[13] Schon P,Schrot G,Wanner G,et al.Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli[J].Fems Microbiol Rev,1995,17(1/2):207-212.
[14] Mader H J,Szostak M P,Hensel A,et al.Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines[J].Vaccine,1997,15(2):195-202.
[15] Eko F O,Mayr U B,Attridge S R,et al.Characterization and immunogenicity of Vibrio cholerae ghosts expressing toxin-coregulated pili[J].J Biotechnol,2000,83(1/2):115-123.
[16] Felnerova D,Kudela P,Bizik J,et al.T cell-specific immune response induced by bacterial ghosts[J].Med Sci Monit,2004,10(10):BR362-370.
[17] Peng W,Si W,Yin L,et al.Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks[J].Immunobiology,2011,216(5):558-565.
[18] Zhao Y M,Jiang X,Qu Y Y,et al.Salmonella detection in powdered dairy products using a novel molecular tool[J].J Dairy Sci,2017,100(5):3480-3496.
[19] Alix E,Miki T,Felix C,et al.Interplay between MgtC and PagC in Salmonella enterica serovar Typhimurium[J].Microb Pathogene-sis,2008,45(3):236-240.
[20] 王宏卫,赵平,张珉,等.鼠伤寒沙门菌pagC启动子的克隆与活性分析[J].微生物学报,2003,43(3):308-314.
[21] 张佳,范欣丽,葛玉梅,等.甲型副伤寒杆菌pagC基因分布及其重组表达产物免疫保护作用[J].浙江大学学报(医学版),2013,42(2):171-176,231.
[22] 智海东,王云峰,陈洪岩.我国兽用基因工程疫苗研发现状与策略[J].动物医学进展,2011,32(6):174-178.
[23] Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proc Natl Acad Sci U S A,2000,97(12):6640-6645.
[24] Murphy K C.Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli[J].J Bacteriol,1998,180(8):2063-2071.
[25] Murphy C N,Fowler R C,Williams A J,et al.Nontyphoidal Salmonella enterica nonsusceptible to both levofloxacin and ceftriaxone in Nebraska,United States 2014-2015[J].Foodborne Pathog Dis,2018,15(4):235-238.
[26] Murphy K C,Campellone K G,Poteete A R.PCR-mediated gene replacement in Escherichia coli[J].Gene,2000,246(1/2):321-330.
[27] Muyrers J P,Zhang Y,Testa G,et al.Rapid modification of bacterial artificial chromosomes by ET-recombination[J].Nucleic Acids Res,1999,27(6):1555-1557.
[28] 王恒,冯尔玲,史兆兴,等.用Red系统快速敲除痢疾杆菌asd基因[J].军事医学科学院院刊,2002(3):172-175.
[29] Wang X,Lu C.Mice orally vaccinate d with Edwardsiella tarda ghosts are significantly protected against infection[J].Vaccine,2009,27(10):1571-1578.