本文選題：隱孢子蟲病 + 間接ELISA�。� 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：作為腹瀉病常見病原之一的隱孢子蟲(Cryptosporidium spp.)能侵入人或動物的腸道,引起隱孢子蟲病(cryptosporidiosis)。在隱孢子蟲的眾多種類中,微小隱孢子蟲(Cryptosporidium parvum)是最常見且危害最大的。對免疫功能發(fā)育不全或缺陷的宿主,隱孢子蟲會造成嚴重的傷害,甚至?xí){生命。目前,隱孢子蟲病防控研究面臨著缺乏敏感、特異、便捷的診斷技術(shù)和有效的治療、免疫預(yù)防方法。因而,篩選免疫原性好、免疫反應(yīng)性高的隱孢子蟲抗原分子,研制敏感、特異的診斷技術(shù)和高保護效果的預(yù)防疫苗是當前隱孢子蟲病研究的關(guān)鍵和重要方向。本研究以重組微小隱孢子蟲CP15/60(r CP15/60)和類鈣調(diào)蛋白(rCML)為抗原,建立了2個隱孢子蟲病間接ELISA診斷方法,并與nested PCR方法進行了比較;利用建立的方法,對從上海地區(qū)3個屠宰場采集的豬田間血清進行了檢測。結(jié)果顯示,兩種重組蛋白的最適包被濃度均為2μg/mL,一抗血清的最佳稀釋度均為1:100,酶標二抗的最佳稀釋度也均為1:800。與nested PCR法檢測結(jié)果比較,rCP15/60-ELISA的陽性和陰性符合率分別為100.0%和80.0%;rCML-ELISA的陽性和陰性符合率分別為66.7%和60.0%。利用2種方法分別對291份田間樣品進行檢測,結(jié)果rCP15/60-ELISA法檢測的陽性率為59.5%,rCML-ELISA法的陽性率為51.5%,前者比后者更敏感。交叉試驗結(jié)果顯示,2種方法對豬附紅細胞體感染豬血清、弓形蟲感染豬血清、豬瘟病毒感染豬血清、豬藍耳病毒感染豬血清的檢測結(jié)果均為陰性。利用rCP15/60-ELISA方法對341份豬田間樣品進行檢測,結(jié)果陽性率為56.89%;利用rCML-ELISA方法對353份豬田間樣品進行檢測,陽性率為44.47%,提示這2種ELISA方法的敏感性較高、特異性較強,可用于實驗室初篩和隱孢子蟲病的流行病學(xué)調(diào)查。為了觀察CP15/60、CML 2種抗原制備的疫苗對小鼠隱孢子蟲病的免疫預(yù)防效果,并與其它部分抗原進行比較,將重組蛋白rCP15/60、rCML,以及rCpT全、rCp Tm、rENO、rP23四個重組蛋白分別與弗氏佐劑充分乳化,通過多次皮下注射的方式免疫小鼠,觀察其體液免疫和細胞免疫應(yīng)答反應(yīng);同時,對小鼠進行泰澤隱孢子蟲卵囊人工感染,觀察免疫小鼠的排卵囊情況。結(jié)果顯示,6個亞單位疫苗免疫試驗組小鼠血清IgG抗體水平、TNF和IL-5細胞因子、脾臟CD4+和CD8+含量都有不同程度提高,說明6組亞單位疫苗都特異性地誘導(dǎo)產(chǎn)生了體液免疫和細胞免疫反應(yīng)。人工感染試驗結(jié)果顯示,rCP15/60免疫組的減卵囊率達45.57%,僅次于rENO免疫組,高于其它各組;rCML免疫組的減卵囊率達34.08%,次于rENO免疫組,與rP23免疫組基本相當,但高于rCpTm和rCpT全免疫組。上述結(jié)果表明,利用rCP15/60和rCML制備的亞單位疫苗對小鼠泰澤隱孢子蟲感染均有較好的免疫保護效果,能誘導(dǎo)宿主產(chǎn)生了顯著的體液和細胞免疫反應(yīng),是隱孢子蟲病預(yù)防疫苗的重要候選抗原,有較高的研究價值。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.9

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本文編號：1831949