Construction and Vaccine Potential of Acapsular Mutants of Erysipelothrix rhusiopathiae: Use of Excision of Tn916 To Inactivate a Target Gene
Construction and Vaccine Potential of Acapsular Mutants of Erysipelothrix rhusiopathiae: Use of Excision of Tn916 To Inactivate a Target Gene
复制标题
猪红斑丹毒丝菌无荚突变体的构建和疫苗潜力：利用 Tn916 切除来灭活靶基因
作者：
Y. Shimoji;Y. Mori;T. Sekizaki;T. Shibahara;Y. Yokomizo
摘要我们以前研究过猪丹毒丝菌Tn 916突变株对小鼠无毒力。在这项研究中，我们构建了非回复无囊突变体，并在小鼠中检测了突变体的疫苗潜力。将代表性的无囊转座子突变体33 H6接种于含有高压灭菌的金霉素和喹哪地酸的选择性琼脂上，获得了两个四环素敏感突变体。Tn 916侧翼突变体的染色体区域的序列分析显示，Tn 916已经自发地从该区域切除，并且推测与Tn 916一起插入33 H6染色体的六个新核苷酸取代了插入位点处存在的核苷酸。通过Western免疫印迹证实突变体不含荚膜抗原，并且对小鼠无毒力（皮下50%致死剂量[LD 50]，>109 CFU）。用一株突变株YS-1进一步研究了无囊突变株用于活疫苗的安全性和有效性。皮下（s.c.）接种。小鼠皮下免疫用2 × 10 ~ 4 ~ 2 × 108 CFU YS-1株免疫小鼠，21天后可完全抵抗100 LD_（50）的同源强毒株Fujisawa-SmR的攻击，且2 × 108 CFU YS-1株免疫小鼠所产生的保护性免疫可持续3个月。在被动免疫实验中，在免疫后第14天和第21天从用菌株YS-1免疫的小鼠收集的血清提供了对Fujisawa-SmR攻击的保护，而在第4天和第7天收集的血清没有。此外，还观察到脾细胞对E.在用菌株YS-1免疫的小鼠中观察到rhusiopathiae抗原，并且在小鼠中免疫后7天观察到针对抗原性异源细菌单核细胞增多性李斯特菌的交叉保护，表明细胞介导的免疫已经被诱导。这些结果表明，E.因此，猪红斑病YS-1疫苗可能是进一步研究疫苗效力的合适选择。
ABSTRACT We previously showed that acapsular transposon Tn916mutants of Erysipelothrix rhusiopathiae are avirulent for mice. In this study, we constructed nonreverting acapsular mutants and examined the vaccine potential of the mutants in mice. A representative acapsular transposon mutant, 33H6, was plated on selective agar containing autoclaved chlortetracycline and quinaldic acid, and two tetracycline-sensitive mutants were obtained. Sequence analysis of chromosomal regions of the mutants in which Tn916 had flanked revealed that Tn916 had spontaneously excised from the region and that the six new nucleotides, which were presumably inserted with Tn916 into 33H6 chromosome, substituted for those present at the insertion site. The mutants were confirmed to be devoid of capsular antigen by Western immunoblotting and were nonvirulent for mice (subcutaneous 50% lethal dose [LD50], >109 CFU). The safety and efficacy of acapsular mutants for live vaccines was further studied by using one mutant strain, named YS-1. The YS-1 bacteria were cleared from the skin sites of inoculation, livers, and spleens of the inoculated mice by 7 days after subcutaneous (s.c.) inoculation. Mice immunized s.c. with doses ranging from 2 × 104 to 2 × 108 CFU of strain YS-1 were completely protected against challenge with 100 LD50 of the homologous, highly virulent strain Fujisawa-SmR 21 days postimmunization, and protective immunity conferred by immunization with 2 × 108 CFU of the strain lasted for as long as the 3 months of the observation period. In passive immunization experiments, sera collected from mice immunized with strain YS-1 at days 14 and 21 postimmunization provided protection against challenge with Fujisawa-SmR, whereas sera collected at days 4 and 7 did not. Furthermore, specific spleen cell responses to E. rhusiopathiae antigens were observed in mice immunized with strain YS-1, and cross-protection against the antigenically heterologous bacterium Listeria monocytogenes was observed at 7 days after immunization in the mice, suggesting that cell-mediated immunity had been induced. These results suggest that E. rhusiopathiae YS-1 may be a suitable choice for further studies of vaccine efficacy in swine.
LPS 缺陷的 C3H/HeJ 小鼠中无毒沙门氏菌诱导的细胞免疫。
DOI：
--
发表时间：
1984
期刊：
Journal of immunology (Baltimore, Md. : 1950)
影响因子：
--
作者：
Eisenstein,TK;Killar,LM;Stocker,BA;Sultzer,BM
通讯作者：
Sultzer,BM
DOI：
10.1073/pnas.84.20.7208
发表时间：
1987-10-01
影响因子：
11.1
作者：
RUBENS, CE;WESSELS, MR;KASPER, DL
通讯作者：
KASPER, DL