猪多杀性巴氏杆菌保护性抗原筛选
发布时间：2018-10-31 17:20
【摘要】：多杀性巴氏杆菌是一种能够引起猪肺疫、多杀性巴氏杆菌肺炎的病原,并且也是猪萎缩性鼻炎的主要病原之一。为了能够研制出一种有效的多杀性巴氏杆菌疫苗,本试验筛选了5个位于外膜的基因,这5个基因分别是NlpI、DamB、OmpH、PlpP和FabA。将这5个基因插入至表达载体pET-28a(+)中,成功获得了重组质粒pET28N1pI、 PET28BamD、PET28OmpH、pET28P1pP 和 pET28FabA;通过测序结果,对获得的基因序列进行生物信息学分析,发现克隆获得的基因在不同多杀性巴氏杆菌菌株的相同基因之间的保守性很高。将转化了重组质粒的重组表达菌进行原核表达。结果可知PET28P1pP和pET28FabA蛋白主要以可溶性蛋白的形式存在,而pET28N1pI、 pET28BamD和PET28OmpH主要以包涵体的形式存在。Western Blot结果表明所获得的重组蛋白均具有一定的反应原性。为了研究获得的重组蛋白能否诱导小鼠机体产生高水平的特异性抗体,同时产生的特异性抗体能否为小鼠提供同型的攻毒保护。将纯化的重组蛋白与铝胶佐剂进行混合,制备成了重组亚单位疫苗。随机挑选了Km系雌性小鼠,并使用已制备好的重组亚单位疫苗免疫小鼠。利用间接ELISA法检测实验组小鼠两次免疫后13d的血清抗体水平,结果发现：二免后13d,除了阴性对照组外,其他实验组的小鼠抗体水平都有所升高。二免后14d,用一定剂量的多杀性巴氏杆菌CVCC 439株对所有小鼠进行攻毒。试验表明NlpI、BamD、OmpH、P1pP和FabA蛋白能够为小鼠提供一定的同型保护,其中P1pP和FabA蛋白的保护性最高,达到了66.67%。为了研究这几个蛋白在不同荚膜抗原血清型之间是否具有交叉保护性,随机挑选了KM雌鼠,用制备好的弗氏佐剂疫苗免疫小鼠,二免后14d对免疫小鼠分别攻以一定量的多杀性巴氏杆菌CVCC 444、CVCC 434和CVCC 439株活菌,并观察攻毒保护结果。攻毒保护试验结果表明PlpP蛋白能够提供较好的交叉保护性,另外FabA也能够为小鼠提供较好的交叉保护性,而其他蛋白有的只能提供微弱的交叉保护性。
[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.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
本文编号：2303014
[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.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.28
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