猪霍乱沙门氏菌C78-1株crp缺失株的构建及其生物学特性初步研究
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摘要
猪霍乱沙门氏菌( Salmonella Choleraesuis )是引起仔猪副伤寒的主要病原之一,可引起断奶仔猪大批发病,常因伴发或继发感染其它疾病或治疗不及时而大批死亡,给养猪业造成重大损失。动物生前感染或食品受到污染,可使人发生食物中毒,在公共卫生上也具有十分重要意义。疫苗接种是防控仔猪副伤寒的最适宜策略。在我国,研制仔猪副伤寒活疫苗比较成功的是有关专家将抗原性良好的猪霍乱沙门氏菌强毒株C78-1接种含有醋酸铊的培养基中传数百代后,选出了免疫原性良好的弱毒株C500。但该菌株是经化学方法传代致弱,对其致弱的遗传背景和引起免疫保护的机理目前仍不清楚。另外,该疫苗菌株具有一定的残余毒力,且存在毒力返强的风险。
本研究旨在研制更加安全有效的猪霍乱沙门氏菌疫苗弱毒株,并为进一步将其开发为适于粘膜免疫的口服疫苗活载体奠定一定基础,以crp(cAMP receptor protein,crp)基因作为猪霍乱沙门氏菌C78-1株减毒基因,运用重组自杀性质粒介导细菌的等位交换技术两步法筛选猪霍乱沙门氏菌C78-1株crp基因工程缺失突变株,并对其生物学特性进行了初步研究。主要内容包括:
1.crp基因的克隆与序列分析
参照鼠伤寒沙门氏菌LT2株(GenBank No:AE008859)crp基因序列,从猪霍乱沙门氏菌C78-1基因组中扩增出crp基因。通过测序和序列对比分析显示,与C500株crp基因全序列完全相同,与Genbank上登录的其它全序列已知的3株沙门氏菌crp基因高度保守。
2.重组自杀性质粒pREΔcrp的构建
以猪霍乱沙门氏菌C78-1基因组为模板,分别扩增crp上下游片段并将其克隆入自杀性质粒pRE112上,转化大肠杆菌χ7213,构建含缺失320bp的crp基因重组自杀性质粒pREΔcrp。
3.接合转移与缺失菌株ΔcrpC78-1的筛选鉴定
以转化了重组自杀性质粒pREΔcrp的大肠杆菌χ7213为供体菌,C78-1为受体菌进行接合转移。利用重组自杀性质粒介导细菌基因等位交换技术两步法筛选crp基因缺失突变株,并进行PCR和测序鉴定,以确定缺失菌株ΔcrpC78-1的构建成功。
4.缺失菌株ΔcrpC78-1生物学特性研究
对缺失菌株ΔcrpC78-1表型、遗传稳定性、生长特性及对小鼠的毒力等生物学特性进行了鉴定,结果发现:缺失菌株?crpC78-1失去了利用麦芽糖、蔗糖、果糖与甘露醇等碳源的能力,不能分解H2S,但保留了利用葡萄糖的能力;血清型与亲本菌株C78-1一致,且能够稳定遗传缺失的crp基因;小鼠致死性试验结果表明其毒力较C78-1降低约750倍。
Salmonella choleraesuis is one of the main pathogens of piglet’s paratyphoid, and it affect mainly weaned piglets. A large number of weaned piglets can be dead when secondary infection with other pathgens, or not treated in time. In addition, this pathogen play a significant role in public health for causing human food poisoning through the infected animals or the contaminated products. Vaccination against S. choleraesuis is an appropriate strategy for control and prevention of this disease. In China, the most effective attenuated paratyphoid fever vaccine strain was C500 strain. It was attenuated from highly virulent S. choleraesuis C78-1 strain by maintaining in the thallium acetate's culture medium for hundreds of generations, and had stronger immunogenicity. However, the genetic background and the mechanism of immune protection of C500 strain attenuated by chemical methods remain still unknown. In addition, the vaccine strain remains some residual virulence and risk of back mutation.
In the current study, a safer attuanted S. choleraesuis vaccine was developed through deleting the crp gene, which coding a cAMP receptor protein. The ?crpC78-1 mutant was constructed through the two-step method introduced by the transduction of recombinant suicide plasmid. The biological characteristics of the ?crpC78-1 was further analysed. The main research was described as follows:
1. Cloning and sequential analysis of crp gene
The crp gene were cloned from genomic DNA of C78-1 strain according to the corresponding sequences of S. typhimurium LT2 strain (AE008859) from GenBank. The results of sequence alignment analysis showed that the C78-1 crp gene was completely same as C500 strain’s and highly conserved compared with other three salmonella whose whole genome sequences were published.
2. Construction of recombination suicide plasmids pREΔcrp
The upstream and downstream fragements of crp gene were amplified from genomic DNA of S. choleraesuis C78-1, then successfully subcloned into suicide plasmid pRE112, transformed into E. coliχ7213. The recombination suicide plasmid were designated as pREΔcrp which contained 320bp-deleted crp fragement.
3. Transconjugation and identification of theΔcrpC78-1 mutant
The E. coil donor strainχ7213 transformed with recombination suicide plasmid pREΔcrp was conjugated with the recipient S. choleraesuis C78-1 strain. The ?crp mutant of S. choleraesuis C78-1 was constructed by the allelic exchange introduced by the transduction of the recombination suicide plasmid. Then, theΔcrpC78-1 mutant was determined by PCR and sequencing analysis.
4. Analysis for Biological Characteristics ofΔcrpC78-1 mutant
The phenotypes, genetic stability, growth properties and virulence in mice ofΔcrpC78-1 mutant were characterized. The O and H antigens of the mutant was 6,7:C:1,5, identical to the parent C78-1 strain. TheΔcrpC78-1 mutant was able to ferment glucose, not maltose, mannose and xylose, while the parental strain C78-1 did. The mutant was stable with the recombinant ?crp gene in vitro. However, fermentation patterns and growth rate of the mutant were differed from the parent C78-1 strain obviously. The mouse lethal test showed that the virulence of the muntant was lower 750 times than C78-1.
本研究旨在研制更加安全有效的猪霍乱沙门氏菌疫苗弱毒株,并为进一步将其开发为适于粘膜免疫的口服疫苗活载体奠定一定基础,以crp(cAMP receptor protein,crp)基因作为猪霍乱沙门氏菌C78-1株减毒基因,运用重组自杀性质粒介导细菌的等位交换技术两步法筛选猪霍乱沙门氏菌C78-1株crp基因工程缺失突变株,并对其生物学特性进行了初步研究。主要内容包括:
1.crp基因的克隆与序列分析
参照鼠伤寒沙门氏菌LT2株(GenBank No:AE008859)crp基因序列,从猪霍乱沙门氏菌C78-1基因组中扩增出crp基因。通过测序和序列对比分析显示,与C500株crp基因全序列完全相同,与Genbank上登录的其它全序列已知的3株沙门氏菌crp基因高度保守。
2.重组自杀性质粒pREΔcrp的构建
以猪霍乱沙门氏菌C78-1基因组为模板,分别扩增crp上下游片段并将其克隆入自杀性质粒pRE112上,转化大肠杆菌χ7213,构建含缺失320bp的crp基因重组自杀性质粒pREΔcrp。
3.接合转移与缺失菌株ΔcrpC78-1的筛选鉴定
以转化了重组自杀性质粒pREΔcrp的大肠杆菌χ7213为供体菌,C78-1为受体菌进行接合转移。利用重组自杀性质粒介导细菌基因等位交换技术两步法筛选crp基因缺失突变株,并进行PCR和测序鉴定,以确定缺失菌株ΔcrpC78-1的构建成功。
4.缺失菌株ΔcrpC78-1生物学特性研究
对缺失菌株ΔcrpC78-1表型、遗传稳定性、生长特性及对小鼠的毒力等生物学特性进行了鉴定,结果发现:缺失菌株?crpC78-1失去了利用麦芽糖、蔗糖、果糖与甘露醇等碳源的能力,不能分解H2S,但保留了利用葡萄糖的能力;血清型与亲本菌株C78-1一致,且能够稳定遗传缺失的crp基因;小鼠致死性试验结果表明其毒力较C78-1降低约750倍。
Salmonella choleraesuis is one of the main pathogens of piglet’s paratyphoid, and it affect mainly weaned piglets. A large number of weaned piglets can be dead when secondary infection with other pathgens, or not treated in time. In addition, this pathogen play a significant role in public health for causing human food poisoning through the infected animals or the contaminated products. Vaccination against S. choleraesuis is an appropriate strategy for control and prevention of this disease. In China, the most effective attenuated paratyphoid fever vaccine strain was C500 strain. It was attenuated from highly virulent S. choleraesuis C78-1 strain by maintaining in the thallium acetate's culture medium for hundreds of generations, and had stronger immunogenicity. However, the genetic background and the mechanism of immune protection of C500 strain attenuated by chemical methods remain still unknown. In addition, the vaccine strain remains some residual virulence and risk of back mutation.
In the current study, a safer attuanted S. choleraesuis vaccine was developed through deleting the crp gene, which coding a cAMP receptor protein. The ?crpC78-1 mutant was constructed through the two-step method introduced by the transduction of recombinant suicide plasmid. The biological characteristics of the ?crpC78-1 was further analysed. The main research was described as follows:
1. Cloning and sequential analysis of crp gene
The crp gene were cloned from genomic DNA of C78-1 strain according to the corresponding sequences of S. typhimurium LT2 strain (AE008859) from GenBank. The results of sequence alignment analysis showed that the C78-1 crp gene was completely same as C500 strain’s and highly conserved compared with other three salmonella whose whole genome sequences were published.
2. Construction of recombination suicide plasmids pREΔcrp
The upstream and downstream fragements of crp gene were amplified from genomic DNA of S. choleraesuis C78-1, then successfully subcloned into suicide plasmid pRE112, transformed into E. coliχ7213. The recombination suicide plasmid were designated as pREΔcrp which contained 320bp-deleted crp fragement.
3. Transconjugation and identification of theΔcrpC78-1 mutant
The E. coil donor strainχ7213 transformed with recombination suicide plasmid pREΔcrp was conjugated with the recipient S. choleraesuis C78-1 strain. The ?crp mutant of S. choleraesuis C78-1 was constructed by the allelic exchange introduced by the transduction of the recombination suicide plasmid. Then, theΔcrpC78-1 mutant was determined by PCR and sequencing analysis.
4. Analysis for Biological Characteristics ofΔcrpC78-1 mutant
The phenotypes, genetic stability, growth properties and virulence in mice ofΔcrpC78-1 mutant were characterized. The O and H antigens of the mutant was 6,7:C:1,5, identical to the parent C78-1 strain. TheΔcrpC78-1 mutant was able to ferment glucose, not maltose, mannose and xylose, while the parental strain C78-1 did. The mutant was stable with the recombinant ?crp gene in vitro. However, fermentation patterns and growth rate of the mutant were differed from the parent C78-1 strain obviously. The mouse lethal test showed that the virulence of the muntant was lower 750 times than C78-1.
引文
[1]徐引弟,郭爱珍,陈焕春.猪霍乱沙门氏菌的危害与防制[J].中国畜牧兽医,2008,35(3):115-117.
[2]蔡宝祥主编.家畜传染病学[M].第三版.北京:中国农业出版社,2001,198-201.
[3] Manzano M, Locolin L, Astori G, et al. Development of a PCR microplate-capture hybridiza- tion method for simple, fast and sensitive detection of salmonella serovars in food[J]. Molecu- lar and ceullar Probes, 1998, 12(4):227-234.
[4]李怀玉.一起猪霍乱沙门氏菌引起食物中毒的调查报告[J].中国医药指南,2008,5(6):86-87.
[5]王俊智,仲冬梅.从上臂脓液中分离1株猪霍乱沙门氏菌[J].工企医刊,2005,18(3):48.
[6]关平,张大东,陈跃光等.人猪霍乱沙门氏茵败血症1例[J].中国疗养医学,2008,17 (4):244-245.
[7] Chiu CH, Chuang CH, Chiu S, et al. Salmonella enterica serotype Choleraesuis infections in pediatric patients[J]. Pediatrics, 2006, 117(6):1193-1196.
[8]贾爱卿,郭爱珍,刘维红等.致病性猪源鼠伤寒沙门氏菌的耐药特征鉴定及耐药性消除研究[J].微生物学报,2006,46(5):789-795.
[9] Smith HW. The immunization of mice, calves, and pigs against Salmonella dublin and Salmo- nella choleraesuis infections[J]. J Hyg, 1965, 63(5):117-135.
[10]徐引第.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.
[11]陆承平主编.兽医微生物学[M].第三版.北京:中国农业出版社,2001,223-229.
[12] Cloak O M, Duffy G, Sheridan J, et al. Development of a surface adhesion immuneo fluores- cent technique for the rapid detection of Salmonella spp from meat and poultry[J]. Journal of Applied Microbiology, 1999, 86(4):583-590.
[13]徐引弟,郭爱,陈焕春等.减毒猪霍乱沙门氏菌作为疫苗及载体的研究进展[J].猪与禽,2007,27(6):71-74.
[14] Karaolis DK, Johnson JA, Bailey CC, et al. A Vibrio cholerae pathogenicity island asso- ciated with epidemic and pandemic strains[J]. ProcNatl Acad Sci, 1998, 95(6):3134-3139.
[15] Terakado, Serizakl, Hashimoto, et al. Correlation between the presenc of a fifty Megadalton plasmid in Salmonella Dublin and virulence for mice[J]. Infection and Immunity, 1983, 41(1):443-444.
[16] Hacker J, Blun-Oehler G, Muhldorfer I, et a1. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution[J]. Mol Microbiol, 1997, 23(6):1089-1097.
[17] Groisman EA, Ochman H. Pathogenicity island: bacterial evolution in quantum leaps[J]. Cell, 1996, 87(5):791-794.
[18] Hueck CJ. TypeⅢprotein secretion systems in bacterial pathogens of animals and plants[J]. Microbiol Mol Biol Rev, 1998, 62(2):379-433.
[19] Zhao S, Qaiyumi S, Friedman S, et al. Characterization of Salmonella enterica Serotype Newport Isolated from Humans and Food Animals[J]. J Clin Microbiol, 2003, 41(12):5366- 5371.
[20] Cardona-Castro N, Restrepo-Pineda E, Correa-Ochoa M. Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica[J]. MemInst Oswal-do Cruz, 2002, 97(8): 1153-1156.
[21] Lichtensteiger CA, Vimr ER. Systemicand enteric colonization of pigs by a hilA signature tag- ged mutant of Salmonella choleraesuis[J]. Microb Pathog, 2003, 34 (3):149-154.
[22] Kimbrough TG, Miller SI. Contribution of Salmonella typhimurium type III secretion components to needle complex formation[J]. Proc Natl Acad Sci, 2000, 97(20):11008-11013.
[23] Nassib TA , El-Din MZ, El-Sharoud WM. Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media[J]. Lett Appl Microbiol, 2003, 37 (5): 405–409.
[24] Pangloli P, Dje Y, Oliver SP, et al. Evaluation of methods for recovery of Salmonella from dairy cattle, poultry, and swine farms[J]. J Food Prot, 2003, 66(11):1987-1995.
[25] Valentin-Bon IE, Brackett RE, Seo KH, et al. Preenrichment versus direct selective agar plating for the detection of Salmonella enteritidis in shell eggs[J]. J Food Prot, 2003, 66 (9): 1670-1674.
[26] Massi MN, Shirakawa T, Gotoh A, et al. Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi[J]. J Infect Chemother, 2003, 9(3): 233-2371.
[27] Malorny B, Hoorfar J, Bunge C, et al. Multicenter validation of the analytical accuracy of Salmonella PCR:towards an international standard[J]. App Environ Microbiol, 2003, 69(1): 290-296.
[28] Shearer AEH, Strapp CM, Joerger RD. Evaluation of polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeriamonocytogenes on fresh fruits and vegetables[J]. J Food Prot, 2001, 64(6):788-795.
[29]向雪菲,刘斌,张利等.食品中沙门氏菌分子检测靶点的筛选与评价[J].微生物学报,2008,48(7):941-946.
[30] Rahn K, De Grandis SA, Clarke RC, et al. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J]. Mol Cell Probes, 1992, 6(4):271-279.
[31] Cocolin L, Manzano M, Cantoh C, et al. Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food[J]. Journal Applied Microbiology, 1998, 85(4):673-677.
[32]陈金顶,索青利,廖明等.沙门氏菌的invA基因序列分析与分子检测[J].中国人兽共患病杂志,2004,20(10):868-871.
[33]陈弟诗,郭万柱,徐志文等.猪霍乱沙门氏菌的分离与鉴定以及PCR检测方法的建立[J].安徽农业科学,2007,35(20):6020-6023.
[34] Hara-Kudo Y, Yoshino M, Kojima T, et al. Loop-mediated isothermal amplificaion for the rapid detection of Salmonella[J]. Fems Microbiol Lett, 2005, 253(1): 155-161.
[35]王敏雅,徐明汉,潘宏伟等.沙门菌invA基因LAMP快速检测法的建立和初步应用[J].中国卫生检验杂志,2008,18(10):1971-1973.
[36] Krysinski EP, Heimsch RC. Use of enzyme-labeled antibodies to detect Salmonella in foods[J]. Appl Environ Microbiol, 1977, 33(4):947-954.
[37] Luk JM, Lindberg AA. Rapid and sensitive detection of Salmonella (O:6,7) by immuneomag- netic monoclonal antibody-based assays[J]. Immunol Methods, 1991, 137(1):1-8.
[38] Torensma R, Visser MJ, Aarsman CJ, et al. Monoclonal antibodies that detect live Salmonella [J]. American Society for Microbiology, 1992, 58(12):3868-3872.
[39]焦新安,王志亮.单克隆抗体测定沙门氏菌鞭毛蛋白属共同抗原表位的分布特性[J].细胞与分子免疫学杂志,1995,11(3):4-10.
[40]殷月兰,潘志,孟书霞,等.单抗竞争ELISA检测猪沙门氏菌感染方法的建立与应用[J].中国预防兽医学报,2003,25(1):65-67.
[41] Nowak B, von Müffling T, Chaunchom S, et al. Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique[J]. International Journal of Food Microbiology, 2007, 115(3):259-267.
[42]杨永恒,樊明涛,李瑜,等.猪肉中沙门氏菌的PCR检测[J].食品研究与开发,2009,30(5):128-130.
[43] Yeh KS, Chen TH, Liao, et a1. PCR amplification of the Salmonella typhimurium fimY genesequence to detect the Salmonella species[J]. International Journal of Food Microbiology, 2002, 78(3):227-234.
[44]张红见,韩志辉,魏建华等.Dot-ELISA检测猪酮体中沙门氏菌的研究[J].黑龙江畜牧兽医,2003(11):8-9.
[45]王友信.LPS-ELISA确定沙门氏菌感染群方法的建立[J].中国禽业导刊,2002,19(18):14-15.
[46] Carmen FR, Jacqueline LH, Altweg GM. Development and evaluation of a broad-range PCR-ELISA assay with Borrelia burgdorferi and Streptococcus pneumoniae as model organisms for reactive arthritis and bacterial meningitis[J]. J Microbiol Meth, 2000, 40(1):79-88.
[47] Wilson T, Carson J, Bowman J. Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA[J]. J Microbiol Meth, 2002, 51(2):163-170.
[48] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. J Mol Endocrinol, 2000, 25(2):169-193.
[49] Daly P, Coller T, Doyle S. PCR-ELISA detection of Escherichia coli in milk[J]. Lett Appl Microbiol, 2002(34):222-226.
[50] Arya M , Shergill IS , Williamson M, et al. Basic principles of real-time quantitative PCR[J]. Expert Rev Mol Diagn, 2005, 5(2):209-219.
[51] Fitts R, Diamond M, Hamilton C, et al. DNA-DNA hybridization assay for detection of Salmonella spp. in foods[J]. Appl Environ Microbiol, 1983, 46(5):1146-1151.
[52] Chizhikov V, Rasooly A, Chumakov K, et al. Microarray analysis of microbial virulence factors[J]. Appl Environ Microbiol, 2001, 67(7):3258-3263.
[53] Spreng S, Dietrich G, Weidinger G, et al. Rational design of Salmonella-based vaccination strategies[J]. Methods, 2006, 38(2):133-143.
[54] Matsui K, Arai T. The comparison of cell-mediated immunity induced by immunization with porin, viable cells and killed cells of Salmonella typhimurium Microbiol[J]. Immunol, 1992, 36(3):269-278.
[55] Germanier R, Fuer E. Isolation and characterization of GalE mutant Ty21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine[J]. J Infect Dis, 1975, 131(5):553-558.
[56] Mckenna AJ, Bygraves JA, Maiden MC, et al. Attenuated typhoid vaccine Salmonella typhi Ty21a: fingerprinting and quality control[J]. Microbiology, 1995, 141(8):1993-2002.
[57] Sluzewska M, Truszczynski M. Immunogenic properties of S. choleraesuis for pigs[J]. Pol Arch Weter, 1975, 17(4):599-608.
[58] Hanna J, McCracken R, O'Brien JJ. Evaluation of a live Salmonella choleraesuis vaccine byintranasal challenge[J]. Res Vet Sci, 1979, 26(2):216-219.
[59] Roof MB, Doitchinoff DD. Safety, efficacy and duration of immunity induced in swine by use of an avirulent live Salmonella choleraesuis-containing vaccine[J]. Am J Vet Res, 1995, 56(1): 39-44.
[60] Roof MB, Kramer TT, Roth JA, et al. Characterization of a Salmonella choleraesuis isolate after repeated neutrophil exposure[J]. Am J Vet Res, 1992, 53(8):1328-1332.
[61] Zhanqin Zhao, Yun Xue, Bin Wu, et al. Subcutaneous Vaccination with Attenuated Salmonella enterica Serovar Choleraesuis C500 Expressing Recombinant Filamentous Hemagglutinin and Pertactin Antigens Protects Mice against Fatal Infections with both S. enterica Serovar Choleraesuis and Bordetella bronchiseptica[J]. Infect and immune, 2008, 76(5):2157-2163.
[62] Nnalue NA, Stocker BA. Some galE mutants of Salmonella choleraesuis retain virulence[J]. Infect Immun, 1986, 54(3):635-640.
[63] Edwards MF, Stocker BA. Construction of delta aroA his delta pur strains of Salmonella typhi [J]. Bacteriol, 1988, 170(9):3991-3995.
[64] Nnalue NA, Stocker BA. Tests of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis[J]. Infect Immun, 1987, 55(4):955-962.
[65] Nnalue NA, Stocker BA. Vaccination route, infectivity and thioglycollate broth administrati- on: effects on live vaccine efficacy of auxotrophic derivatives of Salmonella choleraesuis[J]. Microb Pathog, 1989, 7(4):299-310.
[66] Curtiss RⅢ, Nakayama K, Kelly SM. Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo[J]. Immunol Invest, 1989, 18(4):583-596.
[67] Kelly SM, Bosecker BA, Curtiss RⅢ. Characterization and protective properties of attenuate- ed mutants of Salmonella choleraesuis[J]. Infect Immun, 1992, 60(11):4881-4890.
[68] Kennedy MJ, Yancey RJ, Sanchez MS, et al. Attenuation and immunogenicity of Delta cya Delta crp derivatives of Salmonella choleraesuis in pigs[J]. Infect Immun, 1999, 67(9):4628-4636.
[69] Chu CY, Wang SY, Chen ZW, et al. Heterologous protection in pigs induced by a plasmidcur- ed and crp gene-deleted Salmonella choleraesuis live vaccine[J]. Vaccine, 2007,25(41):7031-7040.
[70] Dom?ínguez-Bernal G, Tierrez A, BartoloméA, et al. Salmonella enterica serovar Choleraes-uis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively[J].Veterinary Microbiology, 2008, 130(3):298-311.
[71] Fang XW. The research of piglet paratyphoid attenuated vaccine[J]. Edition of Research Report.China Institute of Veterinary Drug Control, 1978, 4(2):1-11.
[72] Kang K. The live vaccine of piglet paratyphoid[J]. Chinese Journal of Veterinary Drug, 2003, 37(2):49.
[73] Huang CB, Feng WT, Xue MQ, et al. Oral administration of the live vaccine against swine paratyphoid[J]. Scinentia Agricultura Sinica, 1981, 12(6):89-94.
[74]梁雪芽,宋厚辉,江玲丽等.沙门氏菌的基因工程减毒以及在DNA疫苗载体中的应用[J].中国兽医杂志,2002,38(7):35-37.
[75]訾占超,石佩,陈瑞等.减毒鼠伤寒沙门氏菌载体在兽用疫苗中的运用[J].中国兽药杂志,2009,43(5):33-39.
[76] Stabel TJ, Mayfield JE, Morfitt DC, et al. Oral immunization ofmice and swine with an attenuated Salmonella choleraesuis[Δcya-12Δ(crp-cdt)19] mutant containing a recombinant plasmid[J]. Infect Immun, 1993, 61(2):610-618.
[77] Beliavskaia VA, Timofeev IV, Perminova NG, et al. Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins.2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis[J]. Mol Gen Mikrobiol Virusol, 2000, (3):17-21.
[78] Lee EK, Platt R, Kang SG, et al. Chromosomal integration and expression of the Escherichia coli K88 gene cluster in Salmonella entericaser. Choleraesuis strain 54 (SC54)[J]. Veterinary Microbiology, 2001, 83(2):177-183.
[79] Shiau AL, Chen YL, Liao CY, et al. Prothymosinαenhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis [J]. Vaccine, 2001, 19(28):3947-3956.
[80] Shiau AL, Chen CC, Yo YT, et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin alpha[J]. Vaccine, 2005, 23(48):5563-5571.
[81] Lee CH, Wu CL, Shiau AL. Endostatin gene therapy delivered by Salmonella choleraesuis inmurine tumormodels[J]. J GeneMed, 2004, 6(12):1382-1393.
[82] Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor- specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model[J]. Cancer Gene Ther, 2005, 12(2):175-184.
[79]韩文瑜,黎诚耀,梁焕春等.大肠杆菌K88ac与LT(A-B+)抗原基因质粒在猪霍乱沙门氏菌中的表达[J].兽医大学学报,1992,12(1):7-10.
[80]韩文瑜,黎诚耀,侯万文等.接种仔猪副伤寒-大肠杆菌腹泻双价基因工程菌苗妊娠母猪的抗体应答[J].中国畜禽传染病,1993,(3):40-41.
[81]韩国全,郭万柱,林华等.减毒猪霍乱沙门菌C500作为基因疫苗运载体的研究现状[J].猪业科学,2009(6):80-83.
[82]刘明秋,牛晓峰,严维耀等.以减毒猪霍乱沙门菌为载体的抗O型口蹄疫病毒重组活菌苗在家兔体内免疫应答的初步研究[J].复旦学报,2005,44(4):484-489.
[83]乔红伟,孙金福,韩文瑜等.猪霍乱沙门菌载体介导猪瘟病毒DNA免疫研究[J].生物工程学报,2005,21(6):865-870.
[84]陈弟诗.猪霍乱沙门氏菌携带的含APP apxIIA基因的双启动子表达载体pEPR-apxIIA的构建[D].雅安:四川农业大学,2007.
[85]韩国全,郭万柱,孙志勇等.猪霍乱沙门菌S.C500运载TGEV-S与猪IL-15融合基因疫苗的稳定性及免疫安全性[J].中国兽医科学,2008,38(10):871-877.
[86] Cong W, Jin H, Jiang CD, et al. Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine[J]. EDP Sciences, 2010, 41(30):2-13.
[87]徐引弟,郭爱珍,刘维红等.绿荧光蛋白基因在猪霍乱沙门氏菌C500株asd-缺失株平衡致死载体系统中的表达[J].中国兽医学报,2007,27(4):493-496.
[88]赵战勤,徐引弟,吴斌等.猪霍乱沙门氏菌?asdC500株的生物学特性及作为活疫苗表达载体的应用[J].生物工程学报,2009,25(1):29-36.
[89]赵战勤,王臣,丁轲等.表达T+Pm保护性抗原的重组猪霍乱沙门氏菌C500株的构建及其生物学特性[J].微生物学报,2010,50(1):91-97.
[90] Edwards RA, Keller LH, Schifferli DM. Improved allelic exchange vectors and their use to analyze 987p fimbriae gene expression[J]. Gene, 1998, 207(2):149-157.
[91] Ho YK, Charles MD, Steven AT, et al. Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors[J]. Bacteriol, 2002, 184 (1):307-312.
[92] Rosu V, Chadfield MS, Santona A, et al. Effects of crp deletion in Salmonella enterica serotype Gallinarum[J]. Acta Veterinaria Scandinavica, 2007, 49(5):14-17.
[93] Alper MD, Ames BN. Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants[J]. Bacteriol, 1978, 133(1):149-157.
[2]蔡宝祥主编.家畜传染病学[M].第三版.北京:中国农业出版社,2001,198-201.
[3] Manzano M, Locolin L, Astori G, et al. Development of a PCR microplate-capture hybridiza- tion method for simple, fast and sensitive detection of salmonella serovars in food[J]. Molecu- lar and ceullar Probes, 1998, 12(4):227-234.
[4]李怀玉.一起猪霍乱沙门氏菌引起食物中毒的调查报告[J].中国医药指南,2008,5(6):86-87.
[5]王俊智,仲冬梅.从上臂脓液中分离1株猪霍乱沙门氏菌[J].工企医刊,2005,18(3):48.
[6]关平,张大东,陈跃光等.人猪霍乱沙门氏茵败血症1例[J].中国疗养医学,2008,17 (4):244-245.
[7] Chiu CH, Chuang CH, Chiu S, et al. Salmonella enterica serotype Choleraesuis infections in pediatric patients[J]. Pediatrics, 2006, 117(6):1193-1196.
[8]贾爱卿,郭爱珍,刘维红等.致病性猪源鼠伤寒沙门氏菌的耐药特征鉴定及耐药性消除研究[J].微生物学报,2006,46(5):789-795.
[9] Smith HW. The immunization of mice, calves, and pigs against Salmonella dublin and Salmo- nella choleraesuis infections[J]. J Hyg, 1965, 63(5):117-135.
[10]徐引第.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.
[11]陆承平主编.兽医微生物学[M].第三版.北京:中国农业出版社,2001,223-229.
[12] Cloak O M, Duffy G, Sheridan J, et al. Development of a surface adhesion immuneo fluores- cent technique for the rapid detection of Salmonella spp from meat and poultry[J]. Journal of Applied Microbiology, 1999, 86(4):583-590.
[13]徐引弟,郭爱,陈焕春等.减毒猪霍乱沙门氏菌作为疫苗及载体的研究进展[J].猪与禽,2007,27(6):71-74.
[14] Karaolis DK, Johnson JA, Bailey CC, et al. A Vibrio cholerae pathogenicity island asso- ciated with epidemic and pandemic strains[J]. ProcNatl Acad Sci, 1998, 95(6):3134-3139.
[15] Terakado, Serizakl, Hashimoto, et al. Correlation between the presenc of a fifty Megadalton plasmid in Salmonella Dublin and virulence for mice[J]. Infection and Immunity, 1983, 41(1):443-444.
[16] Hacker J, Blun-Oehler G, Muhldorfer I, et a1. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution[J]. Mol Microbiol, 1997, 23(6):1089-1097.
[17] Groisman EA, Ochman H. Pathogenicity island: bacterial evolution in quantum leaps[J]. Cell, 1996, 87(5):791-794.
[18] Hueck CJ. TypeⅢprotein secretion systems in bacterial pathogens of animals and plants[J]. Microbiol Mol Biol Rev, 1998, 62(2):379-433.
[19] Zhao S, Qaiyumi S, Friedman S, et al. Characterization of Salmonella enterica Serotype Newport Isolated from Humans and Food Animals[J]. J Clin Microbiol, 2003, 41(12):5366- 5371.
[20] Cardona-Castro N, Restrepo-Pineda E, Correa-Ochoa M. Detection of hilA gene sequences in serovars of Salmonella enterica subspecies enterica[J]. MemInst Oswal-do Cruz, 2002, 97(8): 1153-1156.
[21] Lichtensteiger CA, Vimr ER. Systemicand enteric colonization of pigs by a hilA signature tag- ged mutant of Salmonella choleraesuis[J]. Microb Pathog, 2003, 34 (3):149-154.
[22] Kimbrough TG, Miller SI. Contribution of Salmonella typhimurium type III secretion components to needle complex formation[J]. Proc Natl Acad Sci, 2000, 97(20):11008-11013.
[23] Nassib TA , El-Din MZ, El-Sharoud WM. Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media[J]. Lett Appl Microbiol, 2003, 37 (5): 405–409.
[24] Pangloli P, Dje Y, Oliver SP, et al. Evaluation of methods for recovery of Salmonella from dairy cattle, poultry, and swine farms[J]. J Food Prot, 2003, 66(11):1987-1995.
[25] Valentin-Bon IE, Brackett RE, Seo KH, et al. Preenrichment versus direct selective agar plating for the detection of Salmonella enteritidis in shell eggs[J]. J Food Prot, 2003, 66 (9): 1670-1674.
[26] Massi MN, Shirakawa T, Gotoh A, et al. Rapid diagnosis of typhoid fever by PCR assay using one pair of primers from flagellin gene of Salmonella typhi[J]. J Infect Chemother, 2003, 9(3): 233-2371.
[27] Malorny B, Hoorfar J, Bunge C, et al. Multicenter validation of the analytical accuracy of Salmonella PCR:towards an international standard[J]. App Environ Microbiol, 2003, 69(1): 290-296.
[28] Shearer AEH, Strapp CM, Joerger RD. Evaluation of polymerase chain reaction-based system for detection of Salmonella enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeriamonocytogenes on fresh fruits and vegetables[J]. J Food Prot, 2001, 64(6):788-795.
[29]向雪菲,刘斌,张利等.食品中沙门氏菌分子检测靶点的筛选与评价[J].微生物学报,2008,48(7):941-946.
[30] Rahn K, De Grandis SA, Clarke RC, et al. Amplification of an invA gene sequence of Salmonella typhimurium by polymerase chain reaction as a specific method of detection of Salmonella[J]. Mol Cell Probes, 1992, 6(4):271-279.
[31] Cocolin L, Manzano M, Cantoh C, et al. Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food[J]. Journal Applied Microbiology, 1998, 85(4):673-677.
[32]陈金顶,索青利,廖明等.沙门氏菌的invA基因序列分析与分子检测[J].中国人兽共患病杂志,2004,20(10):868-871.
[33]陈弟诗,郭万柱,徐志文等.猪霍乱沙门氏菌的分离与鉴定以及PCR检测方法的建立[J].安徽农业科学,2007,35(20):6020-6023.
[34] Hara-Kudo Y, Yoshino M, Kojima T, et al. Loop-mediated isothermal amplificaion for the rapid detection of Salmonella[J]. Fems Microbiol Lett, 2005, 253(1): 155-161.
[35]王敏雅,徐明汉,潘宏伟等.沙门菌invA基因LAMP快速检测法的建立和初步应用[J].中国卫生检验杂志,2008,18(10):1971-1973.
[36] Krysinski EP, Heimsch RC. Use of enzyme-labeled antibodies to detect Salmonella in foods[J]. Appl Environ Microbiol, 1977, 33(4):947-954.
[37] Luk JM, Lindberg AA. Rapid and sensitive detection of Salmonella (O:6,7) by immuneomag- netic monoclonal antibody-based assays[J]. Immunol Methods, 1991, 137(1):1-8.
[38] Torensma R, Visser MJ, Aarsman CJ, et al. Monoclonal antibodies that detect live Salmonella [J]. American Society for Microbiology, 1992, 58(12):3868-3872.
[39]焦新安,王志亮.单克隆抗体测定沙门氏菌鞭毛蛋白属共同抗原表位的分布特性[J].细胞与分子免疫学杂志,1995,11(3):4-10.
[40]殷月兰,潘志,孟书霞,等.单抗竞争ELISA检测猪沙门氏菌感染方法的建立与应用[J].中国预防兽医学报,2003,25(1):65-67.
[41] Nowak B, von Müffling T, Chaunchom S, et al. Salmonella contamination in pigs at slaughter and on the farm: A field study using an antibody ELISA test and a PCR technique[J]. International Journal of Food Microbiology, 2007, 115(3):259-267.
[42]杨永恒,樊明涛,李瑜,等.猪肉中沙门氏菌的PCR检测[J].食品研究与开发,2009,30(5):128-130.
[43] Yeh KS, Chen TH, Liao, et a1. PCR amplification of the Salmonella typhimurium fimY genesequence to detect the Salmonella species[J]. International Journal of Food Microbiology, 2002, 78(3):227-234.
[44]张红见,韩志辉,魏建华等.Dot-ELISA检测猪酮体中沙门氏菌的研究[J].黑龙江畜牧兽医,2003(11):8-9.
[45]王友信.LPS-ELISA确定沙门氏菌感染群方法的建立[J].中国禽业导刊,2002,19(18):14-15.
[46] Carmen FR, Jacqueline LH, Altweg GM. Development and evaluation of a broad-range PCR-ELISA assay with Borrelia burgdorferi and Streptococcus pneumoniae as model organisms for reactive arthritis and bacterial meningitis[J]. J Microbiol Meth, 2000, 40(1):79-88.
[47] Wilson T, Carson J, Bowman J. Optimisation of one-tube PCR-ELISA to detect femtogram amounts of genomic DNA[J]. J Microbiol Meth, 2002, 51(2):163-170.
[48] Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. J Mol Endocrinol, 2000, 25(2):169-193.
[49] Daly P, Coller T, Doyle S. PCR-ELISA detection of Escherichia coli in milk[J]. Lett Appl Microbiol, 2002(34):222-226.
[50] Arya M , Shergill IS , Williamson M, et al. Basic principles of real-time quantitative PCR[J]. Expert Rev Mol Diagn, 2005, 5(2):209-219.
[51] Fitts R, Diamond M, Hamilton C, et al. DNA-DNA hybridization assay for detection of Salmonella spp. in foods[J]. Appl Environ Microbiol, 1983, 46(5):1146-1151.
[52] Chizhikov V, Rasooly A, Chumakov K, et al. Microarray analysis of microbial virulence factors[J]. Appl Environ Microbiol, 2001, 67(7):3258-3263.
[53] Spreng S, Dietrich G, Weidinger G, et al. Rational design of Salmonella-based vaccination strategies[J]. Methods, 2006, 38(2):133-143.
[54] Matsui K, Arai T. The comparison of cell-mediated immunity induced by immunization with porin, viable cells and killed cells of Salmonella typhimurium Microbiol[J]. Immunol, 1992, 36(3):269-278.
[55] Germanier R, Fuer E. Isolation and characterization of GalE mutant Ty21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine[J]. J Infect Dis, 1975, 131(5):553-558.
[56] Mckenna AJ, Bygraves JA, Maiden MC, et al. Attenuated typhoid vaccine Salmonella typhi Ty21a: fingerprinting and quality control[J]. Microbiology, 1995, 141(8):1993-2002.
[57] Sluzewska M, Truszczynski M. Immunogenic properties of S. choleraesuis for pigs[J]. Pol Arch Weter, 1975, 17(4):599-608.
[58] Hanna J, McCracken R, O'Brien JJ. Evaluation of a live Salmonella choleraesuis vaccine byintranasal challenge[J]. Res Vet Sci, 1979, 26(2):216-219.
[59] Roof MB, Doitchinoff DD. Safety, efficacy and duration of immunity induced in swine by use of an avirulent live Salmonella choleraesuis-containing vaccine[J]. Am J Vet Res, 1995, 56(1): 39-44.
[60] Roof MB, Kramer TT, Roth JA, et al. Characterization of a Salmonella choleraesuis isolate after repeated neutrophil exposure[J]. Am J Vet Res, 1992, 53(8):1328-1332.
[61] Zhanqin Zhao, Yun Xue, Bin Wu, et al. Subcutaneous Vaccination with Attenuated Salmonella enterica Serovar Choleraesuis C500 Expressing Recombinant Filamentous Hemagglutinin and Pertactin Antigens Protects Mice against Fatal Infections with both S. enterica Serovar Choleraesuis and Bordetella bronchiseptica[J]. Infect and immune, 2008, 76(5):2157-2163.
[62] Nnalue NA, Stocker BA. Some galE mutants of Salmonella choleraesuis retain virulence[J]. Infect Immun, 1986, 54(3):635-640.
[63] Edwards MF, Stocker BA. Construction of delta aroA his delta pur strains of Salmonella typhi [J]. Bacteriol, 1988, 170(9):3991-3995.
[64] Nnalue NA, Stocker BA. Tests of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis[J]. Infect Immun, 1987, 55(4):955-962.
[65] Nnalue NA, Stocker BA. Vaccination route, infectivity and thioglycollate broth administrati- on: effects on live vaccine efficacy of auxotrophic derivatives of Salmonella choleraesuis[J]. Microb Pathog, 1989, 7(4):299-310.
[66] Curtiss RⅢ, Nakayama K, Kelly SM. Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo[J]. Immunol Invest, 1989, 18(4):583-596.
[67] Kelly SM, Bosecker BA, Curtiss RⅢ. Characterization and protective properties of attenuate- ed mutants of Salmonella choleraesuis[J]. Infect Immun, 1992, 60(11):4881-4890.
[68] Kennedy MJ, Yancey RJ, Sanchez MS, et al. Attenuation and immunogenicity of Delta cya Delta crp derivatives of Salmonella choleraesuis in pigs[J]. Infect Immun, 1999, 67(9):4628-4636.
[69] Chu CY, Wang SY, Chen ZW, et al. Heterologous protection in pigs induced by a plasmidcur- ed and crp gene-deleted Salmonella choleraesuis live vaccine[J]. Vaccine, 2007,25(41):7031-7040.
[70] Dom?ínguez-Bernal G, Tierrez A, BartoloméA, et al. Salmonella enterica serovar Choleraes-uis derivatives harbouring deletions in rpoS and phoP regulatory genes are attenuated in pigs, and survive and multiply in porcine intestinal macrophages and fibroblasts, respectively[J].Veterinary Microbiology, 2008, 130(3):298-311.
[71] Fang XW. The research of piglet paratyphoid attenuated vaccine[J]. Edition of Research Report.China Institute of Veterinary Drug Control, 1978, 4(2):1-11.
[72] Kang K. The live vaccine of piglet paratyphoid[J]. Chinese Journal of Veterinary Drug, 2003, 37(2):49.
[73] Huang CB, Feng WT, Xue MQ, et al. Oral administration of the live vaccine against swine paratyphoid[J]. Scinentia Agricultura Sinica, 1981, 12(6):89-94.
[74]梁雪芽,宋厚辉,江玲丽等.沙门氏菌的基因工程减毒以及在DNA疫苗载体中的应用[J].中国兽医杂志,2002,38(7):35-37.
[75]訾占超,石佩,陈瑞等.减毒鼠伤寒沙门氏菌载体在兽用疫苗中的运用[J].中国兽药杂志,2009,43(5):33-39.
[76] Stabel TJ, Mayfield JE, Morfitt DC, et al. Oral immunization ofmice and swine with an attenuated Salmonella choleraesuis[Δcya-12Δ(crp-cdt)19] mutant containing a recombinant plasmid[J]. Infect Immun, 1993, 61(2):610-618.
[77] Beliavskaia VA, Timofeev IV, Perminova NG, et al. Construction of an expression plasmid vector for accomplishing in vivo delivery of recombinant biologically active proteins.2. Synthesis of HBcAG in a vaccine strain of Salmonella choleraesuis[J]. Mol Gen Mikrobiol Virusol, 2000, (3):17-21.
[78] Lee EK, Platt R, Kang SG, et al. Chromosomal integration and expression of the Escherichia coli K88 gene cluster in Salmonella entericaser. Choleraesuis strain 54 (SC54)[J]. Veterinary Microbiology, 2001, 83(2):177-183.
[79] Shiau AL, Chen YL, Liao CY, et al. Prothymosinαenhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis [J]. Vaccine, 2001, 19(28):3947-3956.
[80] Shiau AL, Chen CC, Yo YT, et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin alpha[J]. Vaccine, 2005, 23(48):5563-5571.
[81] Lee CH, Wu CL, Shiau AL. Endostatin gene therapy delivered by Salmonella choleraesuis inmurine tumormodels[J]. J GeneMed, 2004, 6(12):1382-1393.
[82] Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor- specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model[J]. Cancer Gene Ther, 2005, 12(2):175-184.
[79]韩文瑜,黎诚耀,梁焕春等.大肠杆菌K88ac与LT(A-B+)抗原基因质粒在猪霍乱沙门氏菌中的表达[J].兽医大学学报,1992,12(1):7-10.
[80]韩文瑜,黎诚耀,侯万文等.接种仔猪副伤寒-大肠杆菌腹泻双价基因工程菌苗妊娠母猪的抗体应答[J].中国畜禽传染病,1993,(3):40-41.
[81]韩国全,郭万柱,林华等.减毒猪霍乱沙门菌C500作为基因疫苗运载体的研究现状[J].猪业科学,2009(6):80-83.
[82]刘明秋,牛晓峰,严维耀等.以减毒猪霍乱沙门菌为载体的抗O型口蹄疫病毒重组活菌苗在家兔体内免疫应答的初步研究[J].复旦学报,2005,44(4):484-489.
[83]乔红伟,孙金福,韩文瑜等.猪霍乱沙门菌载体介导猪瘟病毒DNA免疫研究[J].生物工程学报,2005,21(6):865-870.
[84]陈弟诗.猪霍乱沙门氏菌携带的含APP apxIIA基因的双启动子表达载体pEPR-apxIIA的构建[D].雅安:四川农业大学,2007.
[85]韩国全,郭万柱,孙志勇等.猪霍乱沙门菌S.C500运载TGEV-S与猪IL-15融合基因疫苗的稳定性及免疫安全性[J].中国兽医科学,2008,38(10):871-877.
[86] Cong W, Jin H, Jiang CD, et al. Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine[J]. EDP Sciences, 2010, 41(30):2-13.
[87]徐引弟,郭爱珍,刘维红等.绿荧光蛋白基因在猪霍乱沙门氏菌C500株asd-缺失株平衡致死载体系统中的表达[J].中国兽医学报,2007,27(4):493-496.
[88]赵战勤,徐引弟,吴斌等.猪霍乱沙门氏菌?asdC500株的生物学特性及作为活疫苗表达载体的应用[J].生物工程学报,2009,25(1):29-36.
[89]赵战勤,王臣,丁轲等.表达T+Pm保护性抗原的重组猪霍乱沙门氏菌C500株的构建及其生物学特性[J].微生物学报,2010,50(1):91-97.
[90] Edwards RA, Keller LH, Schifferli DM. Improved allelic exchange vectors and their use to analyze 987p fimbriae gene expression[J]. Gene, 1998, 207(2):149-157.
[91] Ho YK, Charles MD, Steven AT, et al. Transduction-mediated transfer of unmarked deletion and point mutations through use of counterselectable suicide vectors[J]. Bacteriol, 2002, 184 (1):307-312.
[92] Rosu V, Chadfield MS, Santona A, et al. Effects of crp deletion in Salmonella enterica serotype Gallinarum[J]. Acta Veterinaria Scandinavica, 2007, 49(5):14-17.
[93] Alper MD, Ames BN. Transport of antibiotics and metabolite analogs by systems under cyclic AMP control: positive selection of Salmonella typhimurium cya and crp mutants[J]. Bacteriol, 1978, 133(1):149-157.