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  本文選題：布魯氏菌　切入點：vjbR　出處：《西南大學》2011年碩士論文　論文類型：學位論文

【摘要】：布魯氏菌病是一種在世界范圍內廣泛流行的人畜共患病,它的流行不僅對社會經濟發(fā)展造成了嚴重損失,而且給人類健康帶來很大威脅。減毒活疫苗是目前預防布魯氏菌病效果最好的疫苗形式,在國內外有廣泛的使用,然而,由于存在毒力殘存、免疫后無法鑒別診斷等缺點,其使用受到限制。為了克服現(xiàn)有疫苗的這些缺點,利用敲除抗原基因的方法構建標記疫苗株,并評價標記株作為候選減毒活疫苗的可行性,已成為尋找理想減毒活疫苗的一種重要策略。 根據文獻報道相關標記疫苗株的研究情況,在實驗室前期研究成果的基礎上,本研究進一步對16M△vjbR作為標記疫苗株的可行性進行了評價。首先采取抗性替換的方法,重新構建了vjbR抗性基因替換株,然后對標記菌株的毒力表型進行了確認,對其保護性和保護機制進行了分析。之后,進行了vjbR的體外克隆、表達,并探討其區(qū)分疫苗免疫與自然感染的能力,建立了鑒別診斷方法。 由于布魯氏菌不含質粒,很多外源質粒均不能在細菌中復制,基于此特點,我們建立了一種快速構建布魯氏菌無痕缺失突變體的方法,并成功構建了vjbR的突變株：將vjbR基因兩側同源臂分別與卡那抗性基因進行PCR融合,得到含有兩側同源臂和抗性基因的突變盒序列,然后利用T載體克隆,將突變盒序列克隆到T載體,將得到的重組載體轉入布魯氏菌中,通過抗性篩選得到vjbR基因被卡那基因替換的缺失標記株16M△vjbR。 用16M△vjbR標記株腹腔接種免疫小鼠,在免疫后的2、4、6周分別采集免疫小鼠血清,監(jiān)測抗體產生,分離小鼠的脾臟,用菌體蛋白刺激,分析免疫后小鼠的細胞免疫,進行免疫保護性研究。實驗中我們采用了疫苗株Rev.1免疫組做為陽性對照,生理鹽水PBS組作為陰性對照。實驗結果顯示,用缺失標記株16M△vjbR免疫的BALB/c小鼠能夠對野生株B.melitensisl 6M產生很好的免疫保護效果。同時,突變株誘導了特異性抗體IgG的分泌。其細胞免疫水平IFN-γ以及IL-10均有分泌。這說明缺失標記株16M△vjbR能夠誘導細胞免疫和體液免疫,并起到明顯的保護作用。 缺失標記株16M△vjbR能否作為候選疫苗還與是否有合適的診斷抗原有關。我們對布魯氏菌重組蛋白vjbR進行了體外表達,分別用缺失標記株16M△vjbR、野生株B.melitensis16M免疫BALB/c小鼠,用免疫的小鼠血清與純化的蛋白反應來評價蛋白作為診斷抗原的可能性。通過Western blot和間接ELISA法對蛋白的免疫原性進行了檢測,結果顯示vjbR能夠作為診斷抗原,可用于區(qū)分疫苗免疫和自然感染。 以上結果表明,16M△vjbR是一個良好的候選減毒活疫苗株,值得進一步研究。
[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- 緯 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.
【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

【參考文獻】

相關期刊論文 前3條

1 尚德秋;布魯氏菌病再度肆虐及其原因[J];中國地方病防治雜志;2001年01期

2 付思美;白耀霞;曲R，

本文編號：1563752