本文選題：腐蹄病 + 壞死梭桿菌　； 參考：《甘肅農(nóng)業(yè)大學》2017年碩士論文

【摘要】：壞死梭桿菌(Fusobacterium necrophorum)是引起綿羊腐蹄病和肝膿腫等疾病的常見病原菌之一,所致疾病給養(yǎng)羊業(yè)造成很大的經(jīng)濟損失。該病原產(chǎn)生的白細胞毒素是分泌毒素最強的毒力因子之一,能抑制中性粒細胞的吞噬作用,為細菌侵入宿主體內(nèi)提供協(xié)助作用,從而導致疾病的發(fā)生。由于白細胞毒素的抗原表位區(qū)較分散,其中相對較集中的2407~2695之間的氨基酸序列還未見報道。因此,本研究擬從綿羊腐蹄病病料中分離壞死梭桿菌并開展白細胞毒素免疫原性的研究,以期為今后制備壞死梭桿菌所致疾病的有效疫苗奠定基礎。(1)嚴格遵循厭氧條件下從病料中分離培養(yǎng)細菌,通過形態(tài)學觀察、PCR擴增和系統(tǒng)發(fā)育樹分析對分離菌株進行檢測。結果顯示:從感染腐蹄病的綿羊蹄部分離出長絲串珠狀結構的菌體,經(jīng)PCR擴增可知該分離株為壞死梭桿菌陽性,從系統(tǒng)發(fā)育樹可知該分離株上傳所得序列(KY808973)與澳大利亞分離株A39A8(JX678850.1)的親緣關系最近,同源性達99%。(2)應用DNA star軟件,依據(jù)壞死梭桿菌白細胞毒素全長基因的抗原表位區(qū)和開放性閱讀框篩選出一段基因,依此設計引物來擴增目的基因,并將其克隆到pET-32a(+)表達載體上獲得重組質(zhì)粒,轉化到表達菌BL21(DE3)中,經(jīng)IPTG誘導獲得蛋白表達,純化出的蛋白通過Western-blot檢測其反應原性。結果表明:重組蛋白主要以可溶性的形式表達,分子量大小約為35 ku,將其命名為lkt A35;Western-blot檢測顯示,重組蛋白可以與全菌滅活苗制備的高免血清結合,具有較好的反應原性。(3)用獲得的重組蛋白與弗氏佐劑乳化后,免疫小鼠3次,制備多克隆抗體,用間接ELISA檢測其抗體效價。結果顯示:二免和三免的抗體效價分別達到1:64 000和1:128 000,表明本試驗獲得的多克隆抗體具有較高的抗體效價。
[Abstract]:Fusobacterium necrophorum (Fusobacterium necrophorum) is one of the common pathogens of sheep hoof rot and liver abscess.The leucocyte toxin produced by the pathogen is one of the most virulent factors secreting toxins, which can inhibit the phagocytosis of neutrophils and provide assistance for bacteria to invade the host body, thus leading to the occurrence of diseases.Due to the dispersion of antigenic epitopes of leukocyte toxins, the amino acid sequences between 2407 and 2695, which are relatively concentrated, have not been reported.Therefore, the aim of this study was to isolate Clostridium Necrosis from sheep hoof rot and to study the immunogenicity of leukocyte toxin.In order to lay the foundation for the preparation of the effective vaccine caused by Clostridium Necrosis in the future, the bacteria were isolated and cultured from the diseased materials under anaerobic condition strictly, and the isolated strains were detected by morphological observation PCR amplification and phylogenetic tree analysis.The results showed that filamentous beads were isolated from the hoof of sheep infected with hoof rot, and the strain was positive for Clostridium necrotizing by PCR amplification.The phylogenetic tree showed that the sequence KY808973) was most closely related to the Australian strain A39A8 (JX678850.1), and the homology was 99%. (2) DNA star software was used.According to the antigen epitope region and open reading frame of the full-length gene of Clostridium necrosin, a gene was screened out, and the target gene was amplified by using this primer and cloned into the expression vector pET-32a () to obtain the recombinant plasmid.The protein was induced by IPTG and the purified protein was detected by Western-blot.The results showed that the recombinant protein was mainly expressed in soluble form, and its molecular weight was about 35 ku. the recombinant protein was named lkt A35 Western-blot. The recombinant protein could bind to the high immune serum prepared by the whole bacterium inactivated vaccine.After emulsified with Freund's adjuvant, mice were immunized for 3 times to prepare polyclonal antibodies. The titers of polyclonal antibodies were detected by indirect ELISA.The results showed that the titers of the second and third immunizations reached 1:64 000 and 1: 128 000 respectively, which indicated that the polyclonal antibodies obtained in this experiment had higher titers.
【學位授予單位】：甘肅農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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