隐孢子虫病免疫学诊断方法的建立和应用及其免疫预防研究

发布时间：2018-05-02 02:10

本文选题：隐孢子虫病 + 间接ELISA　；参考：《中国农业科学院》2015年硕士论文

【摘要】：作为腹泻病常见病原之一的隐孢子虫(Cryptosporidium spp.)能侵入人或动物的肠道,引起隐孢子虫病(cryptosporidiosis)。在隐孢子虫的众多种类中,微小隐孢子虫(Cryptosporidium parvum)是最常见且危害最大的。对免疫功能发育不全或缺陷的宿主,隐孢子虫会造成严重的伤害,甚至会威胁生命。目前,隐孢子虫病防控研究面临着缺乏敏感、特异、便捷的诊断技术和有效的治疗、免疫预防方法。因而,筛选免疫原性好、免疫反应性高的隐孢子虫抗原分子,研制敏感、特异的诊断技术和高保护效果的预防疫苗是当前隐孢子虫病研究的关键和重要方向。本研究以重组微小隐孢子虫CP15/60(r CP15/60)和类钙调蛋白(rCML)为抗原,建立了2个隐孢子虫病间接ELISA诊断方法,并与nested PCR方法进行了比较;利用建立的方法,对从上海地区3个屠宰场采集的猪田间血清进行了检测。结果显示,两种重组蛋白的最适包被浓度均为2μg/mL,一抗血清的最佳稀释度均为1:100,酶标二抗的最佳稀释度也均为1:800。与nested PCR法检测结果比较,rCP15/60-ELISA的阳性和阴性符合率分别为100.0%和80.0%;rCML-ELISA的阳性和阴性符合率分别为66.7%和60.0%。利用2种方法分别对291份田间样品进行检测,结果rCP15/60-ELISA法检测的阳性率为59.5%,rCML-ELISA法的阳性率为51.5%,前者比后者更敏感。交叉试验结果显示,2种方法对猪附红细胞体感染猪血清、弓形虫感染猪血清、猪瘟病毒感染猪血清、猪蓝耳病毒感染猪血清的检测结果均为阴性。利用rCP15/60-ELISA方法对341份猪田间样品进行检测,结果阳性率为56.89%;利用rCML-ELISA方法对353份猪田间样品进行检测,阳性率为44.47%,提示这2种ELISA方法的敏感性较高、特异性较强,可用于实验室初筛和隐孢子虫病的流行病学调查。为了观察CP15/60、CML 2种抗原制备的疫苗对小鼠隐孢子虫病的免疫预防效果,并与其它部分抗原进行比较,将重组蛋白rCP15/60、rCML,以及rCpT全、rCp Tm、rENO、rP23四个重组蛋白分别与弗氏佐剂充分乳化,通过多次皮下注射的方式免疫小鼠,观察其体液免疫和细胞免疫应答反应;同时,对小鼠进行泰泽隐孢子虫卵囊人工感染,观察免疫小鼠的排卵囊情况。结果显示,6个亚单位疫苗免疫试验组小鼠血清IgG抗体水平、TNF和IL-5细胞因子、脾脏CD4+和CD8+含量都有不同程度提高,说明6组亚单位疫苗都特异性地诱导产生了体液免疫和细胞免疫反应。人工感染试验结果显示,rCP15/60免疫组的减卵囊率达45.57%,仅次于rENO免疫组,高于其它各组;rCML免疫组的减卵囊率达34.08%,次于rENO免疫组,与rP23免疫组基本相当,但高于rCpTm和rCpT全免疫组。上述结果表明,利用rCP15/60和rCML制备的亚单位疫苗对小鼠泰泽隐孢子虫感染均有较好的免疫保护效果,能诱导宿主产生了显著的体液和细胞免疫反应,是隐孢子虫病预防疫苗的重要候选抗原,有较高的研究价值。
[Abstract]:Cryptosporidium spp.., one of the common pathogens of diarrhoeal diseases. It invades the intestines of humans or animals and causes cryptosporidiosism. Cryptosporidium parvum is the most common and harmful species of Cryptosporidium. Cryptosporidium may cause serious injury and even threaten life to the host with hypoplasia or deficiency of immune function. At present, Cryptosporidiosis prevention and control research is faced with a lack of sensitive, specific, convenient diagnostic techniques and effective treatment, immune prevention methods. Therefore, screening Cryptosporidium antigens with good immunogenicity and high immunoreactivity, developing sensitive, specific diagnostic techniques and high protective preventive vaccines are the key and important directions in the research of Cryptosporidiosis. In this study, using recombinant Cryptosporidium microsporidium CP15/60(r CP15 / 60) and calmodulin-like rCML) as antigens, two indirect ELISA diagnostic methods for Cryptosporidiosis were established and compared with nested PCR method. The serum samples collected from three slaughterhouses in Shanghai were tested. The results showed that the optimal coating concentration of the two recombinant proteins was 2 渭 g / mL, the optimal dilution of the first antiserum was 1: 100, and the optimal dilution of the enzyme labeled second antibody was 1: 800. Compared with the results of nested PCR assay, the positive and negative coincidence rates of rCP15 / 60-ELISA were 100.0% and 80.0%, respectively. The positive and negative coincidence rates of rCML-ELISA were 66.7% and 60.050%, respectively. Two methods were used to detect 291 field samples. The results showed that the positive rate of rCP15/60-ELISA was 59.5% and the positive rate of rCML-ELISA was 51.5%. The former was more sensitive than the latter. The results of cross test showed that the two methods were negative for porcine serum infected with Eperythrozoon, toxoplasma gondii, swine fever virus and blue ear virus. The rCP15/60-ELISA method was used to detect 341 pig field samples, the positive rate was 56.89, and the positive rate was 44.47 by using rCML-ELISA method in 353 pig field samples, which indicated that the two ELISA methods had higher sensitivity and specificity. It can be used for laboratory screening and epidemiological investigation of Cryptosporidiosis. In order to observe the immunoprophylaxis effect of CP15 / 60CML vaccine against Cryptosporidiosis in mice, and compare with other antigens, the recombinant protein rCP15 / 60rCML and the recombinant protein rCp TmENON rP23 were emulsified with Freund's adjuvant, respectively. The humoral and cellular immune responses of mice were observed by multiple subcutaneous injection, and the oocysts of Cryptosporidium taizei were artificially infected to observe the ovulatory sac of immunized mice. The results showed that the levels of serum IgG antibody and IL-5 cytokines, spleen CD4 and CD8 in 6 subunit vaccine immunized mice were increased in varying degrees. The results showed that all of the 6 subunit vaccines specifically induced humoral and cellular immune responses. The results of artificial infection test showed that the oocyst reduction rate of rCP15 / 60 immunized group was 45.57, second only to that of rENO immunization group, and was higher than that of other groups (34.08%), which was similar to that of rP23 immunization group, but higher than that of rCpTm and rCpT immunization group. The results indicated that the subunit vaccine prepared by rCP15/60 and rCML had a good protective effect on the infection of Cryptosporidium taizei in mice, and could induce the host to produce a significant humoral and cellular immune response. Cryptosporidiosis vaccine is an important candidate antigen, which has high research value.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.9

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