【作者】 栗绍文；

【导师】 姚宝安；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 流产嗜性衣原体(Chlamydophila abortus,CAB)是引起猪、牛、羊等多种动物流产、死胎、弱胎等慢性接触性疾病的一种重要病原体,孕妇也可感染而发生流产。建立准确、快速的诊断方法和研制安全、高效的疫苗是预防和控制本病的重要措施。本研究应用PCR技术获得CAB主要外膜蛋白(MOMP)全长基因和四种不同片段大小多型性外膜蛋白90(POMP90)的编码基因,构建重组表达质粒,进行原核表达获得重组蛋白,探讨了其在猪CAB感染血清学检测中的应用价值,建立了猪CAB间接ELISA抗体检测方法;并构建了momp基因真核表达质粒,及细胞因子IL-2、IFN-γ编码基因与momp基因真核共表达质粒,比较了pcDNA3.1和PCI-neo两种真核表达载体基因免疫效应的差异,探索了细胞因子和中药提取成分枸杞多糖对基因免疫的佐剂效应。1.本研究根据已知基因序列设计引物,以流产嗜性衣原体CP/12株的总DNA为模板,PCR扩增了momp全长基因和四种不同片段大小的pomp90基因,与pMD18-T载体连接后进行测序鉴定。将目的基因与原核表达载体pGEX-KG进行连接,构建重组表达质粒,在大肠杆菌E.coli BL21中实现了高效表达,表达产物经Western-blot检测分析,结果表明具有较强的免疫原性。进而以纯化后的五种重组蛋白作为包被抗原,建立了间接ELISA抗体检测方法,并对临床血清进行了检测。结果表明,重组MOMP蛋白不适合于建立间接ELISA抗体检测方法,而四种重组POMP蛋白建立的间接ELISA抗体检测方法均具有灵敏度高、特异性好、准确等优点,且克服了传统间接血凝试验低灵敏度、判断主观性强等缺点。2.将momp基因分别克隆到真核表达载体pcDNA3.1和PCI-neo,构建真核表达质粒pcDNA3.1-MOMP和PCI-MOMP,并进行了小鼠免疫试验,比较不同真核表达载体构建基因疫苗免疫效应的差异。结果表明,PCI-MOMP可诱导更高的体液免疫效应和微弱的细胞免疫,但未观察到更有效的免疫保护。3.将获得的猪IL-2和IFN-γ编码基因插入真核表达质粒PCI-MOMP中构建真核共表达质粒,并通过小鼠接种试验评价了两种细胞因子通过真核表达质粒混合免疫和基因共表达对基因免疫的佐剂效应。结果表明,两种细胞因子既不能增强体液免疫效应,也未观察到增强的细胞免疫效应和有效的免疫保护。4.将中药提取成分枸杞多糖与真核表达质粒PCI-MOMP同时对小鼠进行免疫,评价了枸杞多糖对基因免疫的佐剂效应。结果表明1,枸杞多糖可显著增强体液免疫效应,但未观察到更高的细胞免疫效应和免疫保护效果。本论文建立了猪流产嗜性衣原体间接ELISA抗体检测方法,探索了momp基因疫苗的免疫效应,以及不同真核表达载体对基因免疫效应的影响,细胞因子和枸杞多糖对momp基因疫苗的免疫佐剂效应,并取得了一定效果,为进一步开展猪流产嗜性衣原体病的免疫预防和诊断研究奠定了基础。更多还原

【Abstract】 Chlamydophila abortus (CAB) is one of the important pathogens causing the abortion of sow, cow and ewe and pregnant women. The establishment of rapid and accurate diagnostic method and the development of safe and efficient vaccine are essential measures for the prevention and control of this disease. In this study, the genes encoding major outer membrane protein (MOMP) and four different fragments of polymorphic outer membrane protein-90 (POMP90) of CAB were obtained by PCR. Applicabilities of the recombinant proteins were explored and the indirect ELISA methods for detecting the CAB antibody in sera of pigs were developed. Then the vaccinal plasmids with CAB momp gene were reconstructed, and such swine cytokines as interleukin-2 (IL-2) and interferon gammar (IFN-γ) encoding genes were individually inserted into the vaccinal plasmid for the double expression. Differences of the immune responses induced by the vaccinal plasmid with pcDNA3.1 and PCI-neo as the expression vector were researched, and the immune adjuvant effects of two cytokines and the lycium bartarum polysaccharides (LBPS) were explored.1. In this study, the full-length momp gene and four different fragments of pomp90 gene were amplified with the specific primers and the total DNA template of CAB strain CP/12. The obtained genes identified by sequencing after linked to the pMD18-T vector were linked to the prokaryotic expression vector pGEX-KG for expression. The recombinant plasmids were high-level expressed in the E.coli BL21. Western-blot of recombinant proteins exhitited strong immunogenicities. The indirect ELISA methods established using the five purified recombinant proteins as coating antigens were clinically used to detect the serum CAB specific antibody. While the recombinant MOMP protein was not suitable for the establishment of the ELISA method, the results showed that the methods developed with the four recombinant POMPs were sensitive, specific, accurate, which can overcome the shortcomings of IHA, such as low sensitivity, subjective judgments.2. Then the momp gene was cloned into the eukaryotic expression vector pcDNA3.1 and PCI-neo to reconstruct the vaccinal plasmids pcDNA3.1-MOMP and PCI-MOMP. The immunities were evaluated in mice model and the efficiency of genetic vaccines with various expression vectors was discussed. The results indicated that PCI-MOMP could induce higher humoral immune and celluar immune than pcDNA-MOMP did, but effective immune protection was not observed.3. To increasing the immunity of momp genetic vaccination, the swine IL-2 and IFN-γencoding genes were individually inserted into the vaccinal plasmid PCI-MOMP. The adjuvant effects of the two cytokines on the genetic vaccination through co-administration and double expression were explored in mice model. The results suggested that both humoral and celluar immune response were not increased by the two cytokines, and effective immune protection failed to be induced.4. The adjuvant effect of LBPS on the momp genetic vaccination by co-administration was also explored in mice model. The results showed that LBPS significantly increased the humoral immune response, however the higher celluar immune response and immune protection were not observed.In this paper, indirect ELISA methods were established for detecting the serum swine CAB antibodies. The immune effects of CAB momp genetic vaccine were explored in mice model, incuding the difference between the plasmids constructed with different expression vector, and the adjuvant effects of two cytokines and LBPS on the genetic vaccination. This study was the basis for the further research on immune prevention and diagnosis of swine Chlamydophila abortus infection.更多还原