【摘要】：多殺性巴氏桿菌是一種能夠引起豬肺疫、多殺性巴氏桿菌肺炎的病原,并且也是豬萎縮性鼻炎的主要病原之一。為了能夠研制出一種有效的多殺性巴氏桿菌疫苗,本試驗篩選了5個位于外膜的基因,這5個基因分別是NlpI、DamB、OmpH、PlpP和FabA。將這5個基因插入至表達載體pET-28a(+)中,成功獲得了重組質粒pET28N1pI、 PET28BamD、PET28OmpH、pET28P1pP 和 pET28FabA;通過測序結果,對獲得的基因序列進行生物信息學分析,發(fā)現(xiàn)克隆獲得的基因在不同多殺性巴氏桿菌菌株的相同基因之間的保守性很高。將轉化了重組質粒的重組表達菌進行原核表達。結果可知PET28P1pP和pET28FabA蛋白主要以可溶性蛋白的形式存在,而pET28N1pI、 pET28BamD和PET28OmpH主要以包涵體的形式存在。Western Blot結果表明所獲得的重組蛋白均具有一定的反應原性。為了研究獲得的重組蛋白能否誘導小鼠機體產生高水平的特異性抗體,同時產生的特異性抗體能否為小鼠提供同型的攻毒保護。將純化的重組蛋白與鋁膠佐劑進行混合,制備成了重組亞單位疫苗。隨機挑選了Km系雌性小鼠,并使用已制備好的重組亞單位疫苗免疫小鼠。利用間接ELISA法檢測實驗組小鼠兩次免疫后13d的血清抗體水平,結果發(fā)現(xiàn)：二免后13d,除了陰性對照組外,其他實驗組的小鼠抗體水平都有所升高。二免后14d,用一定劑量的多殺性巴氏桿菌CVCC 439株對所有小鼠進行攻毒。試驗表明NlpI、BamD、OmpH、P1pP和FabA蛋白能夠為小鼠提供一定的同型保護,其中P1pP和FabA蛋白的保護性最高,達到了66.67%。為了研究這幾個蛋白在不同莢膜抗原血清型之間是否具有交叉保護性,隨機挑選了KM雌鼠,用制備好的弗氏佐劑疫苗免疫小鼠,二免后14d對免疫小鼠分別攻以一定量的多殺性巴氏桿菌CVCC 444、CVCC 434和CVCC 439株活菌,并觀察攻毒保護結果。攻毒保護試驗結果表明PlpP蛋白能夠提供較好的交叉保護性,另外FabA也能夠為小鼠提供較好的交叉保護性,而其他蛋白有的只能提供微弱的交叉保護性。
[Abstract]:Pasteurella multocida is one of the main pathogens of porcine atrophic rhinitis. In order to develop an effective Pasteurella multocida vaccine, five genes located on the outer membrane, NlpI,DamB,OmpH,PlpP and FabA., were screened. The five genes were inserted into the expression vector pET-28a () and the recombinant plasmids pET28N1pI, PET28BamD,PET28OmpH,pET28P1pP and pET28FabA; were successfully obtained. The results of sequencing showed that the cloned genes were highly conserved among the same genes of different Pasteurella multocida strains. The recombinant expression strain transformed into the recombinant plasmid was expressed in prokaryotic. The results showed that PET28P1pP and pET28FabA existed mainly in the form of soluble proteins, while pET28N1pI, pET28BamD and PET28OmpH existed. Western Blot in the form of inclusion bodies. The results showed that the recombinant proteins obtained had certain reactivity. In order to study whether the recombinant protein can induce the mice to produce high level of specific antibodies, and whether the specific antibodies can provide the same type of protection for mice. The recombinant subunit vaccine was prepared by mixing the purified recombinant protein with aluminum gel adjuvant. Km female mice were randomly selected and immunized with recombinant subunit vaccine. Indirect ELISA method was used to detect the serum antibody level of the mice in the experimental group on the 13th day after twice immunization. The results showed that the antibody level of the other experimental groups was increased except the negative control group on the 13th day after the second immunization. All mice were challenged with a certain dose of Pasteurella multocida strain CVCC 439 on the 14th day after the second immunization. The results showed that NlpI,BamD,OmpH,P1pP and FabA proteins could provide the same type of protection for mice, and P1pP and FabA proteins were the most protective, reaching 66.67. In order to study whether these proteins have cross-protection between different capsule antigen serotypes, KM female mice were randomly selected and immunized with a prepared Freund's adjuvant vaccine. The mice were immunized with a certain amount of CVCC 444CVCC434 and CVCC 439 live bacteria on the 14th day after the second immunization, and the protective effect was observed. The results showed that PlpP protein could provide better cross-protection, and FabA could provide better cross-protection for mice, while some other proteins could provide only weak cross-protection.
【學位授予單位】：湖南農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.28

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