布鲁氏菌vjbR基因标记疫苗株的构建、免疫保护性分析及鉴别诊断研究

发布时间：2018-03-04 02:26

本文选题：布鲁氏菌　切入点：vjbR　出处：《西南大学》2011年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌病是一种在世界范围内广泛流行的人畜共患病,它的流行不仅对社会经济发展造成了严重损失,而且给人类健康带来很大威胁。减毒活疫苗是目前预防布鲁氏菌病效果最好的疫苗形式,在国内外有广泛的使用,然而,由于存在毒力残存、免疫后无法鉴别诊断等缺点,其使用受到限制。为了克服现有疫苗的这些缺点,利用敲除抗原基因的方法构建标记疫苗株,并评价标记株作为候选减毒活疫苗的可行性,已成为寻找理想减毒活疫苗的一种重要策略。根据文献报道相关标记疫苗株的研究情况,在实验室前期研究成果的基础上,本研究进一步对16M△vjbR作为标记疫苗株的可行性进行了评价。首先采取抗性替换的方法,重新构建了vjbR抗性基因替换株,然后对标记菌株的毒力表型进行了确认,对其保护性和保护机制进行了分析。之后,进行了vjbR的体外克隆、表达,并探讨其区分疫苗免疫与自然感染的能力,建立了鉴别诊断方法。由于布鲁氏菌不含质粒,很多外源质粒均不能在细菌中复制,基于此特点,我们建立了一种快速构建布鲁氏菌无痕缺失突变体的方法,并成功构建了vjbR的突变株：将vjbR基因两侧同源臂分别与卡那抗性基因进行PCR融合,得到含有两侧同源臂和抗性基因的突变盒序列,然后利用T载体克隆,将突变盒序列克隆到T载体,将得到的重组载体转入布鲁氏菌中,通过抗性筛选得到vjbR基因被卡那基因替换的缺失标记株16M△vjbR。用16M△vjbR标记株腹腔接种免疫小鼠,在免疫后的2、4、6周分别采集免疫小鼠血清,监测抗体产生,分离小鼠的脾脏,用菌体蛋白刺激,分析免疫后小鼠的细胞免疫,进行免疫保护性研究。实验中我们采用了疫苗株Rev.1免疫组做为阳性对照,生理盐水PBS组作为阴性对照。实验结果显示,用缺失标记株16M△vjbR免疫的BALB/c小鼠能够对野生株B.melitensisl 6M产生很好的免疫保护效果。同时,突变株诱导了特异性抗体IgG的分泌。其细胞免疫水平IFN-γ以及IL-10均有分泌。这说明缺失标记株16M△vjbR能够诱导细胞免疫和体液免疫,并起到明显的保护作用。缺失标记株16M△vjbR能否作为候选疫苗还与是否有合适的诊断抗原有关。我们对布鲁氏菌重组蛋白vjbR进行了体外表达,分别用缺失标记株16M△vjbR、野生株B.melitensis16M免疫BALB/c小鼠,用免疫的小鼠血清与纯化的蛋白反应来评价蛋白作为诊断抗原的可能性。通过Western blot和间接ELISA法对蛋白的免疫原性进行了检测,结果显示vjbR能够作为诊断抗原,可用于区分疫苗免疫和自然感染。以上结果表明,16M△vjbR是一个良好的候选减毒活疫苗株,值得进一步研究。
[Abstract]:Brucellosis is a widespread zoonosis in the world, which not only causes serious loss to social and economic development. The attenuated live vaccine is currently the most effective vaccine against brucellosis and is widely used at home and abroad. However, because of its virulence, it is unable to differentiate diagnosis after immunization. Its use is limited. In order to overcome these shortcomings of existing vaccines, the tagged vaccine strain is constructed using the method of knockout of antigen genes and the feasibility of using the marker strain as a candidate live attenuated vaccine is evaluated. It has become an important strategy to find an ideal live attenuated vaccine. On the basis of the previous research results, the feasibility of 16m vjbR as a marker vaccine strain was evaluated. First, the method of resistance substitution was adopted. The vjbR resistance gene replacement strain was reconstructed, the virulence phenotype of the labeled strain was confirmed, and its protective and protective mechanisms were analyzed. After that, the vjbR was cloned and expressed in vitro. The ability of distinguishing vaccine immunity from natural infection was discussed, and the differential diagnosis method was established. Because Brucella does not contain plasmids, many foreign plasmids can not be duplicated in bacteria. The mutants of vjbR were successfully constructed: the vjbR gene was fused with the kana-resistant gene by PCR fusion, and the sequence of the cassette containing the two homologous arms and the resistance gene was obtained, and then the T-vector was used to clone the mutants. The mutant box sequence was cloned into T vector, and the recombinant vector was transferred into Brucella. The deletion marker strain 16m vjbRof vjbR gene was obtained by resistance screening. The immunized mice were inoculated with 16m vjbR labeled strain intraperitoneally. The sera of the immunized mice were collected for 6 weeks after immunization, the antibody production was monitored, the spleen of the mice was isolated, and the cellular immunity of the immunized mice was analyzed by the stimulation of the bacterial protein. We used the vaccine strain Rev.1 as the positive control group and the saline PBS group as the negative control. The BALB/c mice immunized with 16M vjbR were able to protect the wild strain B.melitensis l6M. At the same time, The mutant induced the secretion of specific antibody IgG, and its cellular immune level IFN- 纬 and IL-10 were secreted, which indicated that the deletion labeled strain 16M vjbR could induce cellular immunity and humoral immunity, and play an obvious protective role. The recombinant protein vjbR of brucella was expressed in vitro and immunized BALB/c mice with deletion marker strain 16m vjbR and wild strain B.melitensis 16M respectively. The immunogenicity of protein was detected by Western blot and indirect ELISA. The results showed that vjbR could be used as diagnostic antigen. It can be used to distinguish vaccine immunity from natural infection. The above results indicate that 16 M vjbR is a good candidate attenuated live vaccine strain, which deserves further study.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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