【作者】 任文陟；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2010， 博士

【摘要】 隐孢子虫(Cryptosporidium.spp)是寄生于人类及脊椎动物的呼吸道或消化道上皮细胞的一种重要原虫,由它引起的在临床上表现为严重水样腹泻症状的疾病称为隐孢子虫病。1907年,Tyzzer首次报道了隐孢子虫感染小鼠的病例,直到1982年,美国疾病控制中心报道了来自美国六个城市的21名AIDS男性患者由于隐孢子虫病引起了严重的腹泻,才引起全世界研究隐孢子虫的热潮,隐孢子虫病已经被列为美国六大腹泻病之一。2003年我国科技部把隐孢子虫病列入“新发传染病等防治和应用”重大科技攻关项目中,而且是仅有的两种寄生虫病之一。隐孢子虫到目前为止已发现20种以上,但大多数科学家认为其中只有19种是有效种,安氏隐孢子虫(Cryptosporidium andersoni)为其中之一。2000年安氏隐孢子虫被正式命名,其主要感染育成牛和成年牛,奶牛感染后可以引起产奶量下降,体重减轻,是影响奶牛业发展的重要致病因素之一。近年来,Leoni和Morse分别在人体内鉴定出安氏隐孢子虫的存在。虽然国内外对隐孢子虫病已研究多年,但尚无有效的预防办法及防治药物,已筛选了200多种药物,包括所有的抗生素、抗球虫药和磺胺类药物用于该病的治疗,但没有任何药物对隐孢子虫有杀灭作用,因此免疫预防成为控制该病的主要途径,而其关键在于筛选到有效的抗隐孢子虫疫苗的候选抗原。安氏隐孢子虫是一种寄生性原虫,人也有感染的报道。目前,国内外对其研究主要集中在流行病学调查方面,疫苗候选基因的筛选和交叉保护性的研究未见报道。隐孢子虫病与宿主的免疫系统关系密切。对该病的治疗和预防,抗体和疫苗将起重要作用,获得安氏隐孢子虫疫苗的候选基因及交叉保护性研究将为隐孢子虫病的防治提供新的研究方向。筛选免疫相关基因的方法很多,通过文库筛选免疫蛋白相关基因是一个比较普遍的技术方法,噬菌体展示技术是一种可用于筛选新抗原基因的生物技术,此项技术已经被广泛地应用在生命科学研究的各项领域中。通过T7噬菌体展示文库筛选免疫相关基因在安氏隐孢子虫上的应用未见报道。2003年,Andre等通过实验证明安氏隐孢子虫可以成功体外感染人和牛的上皮细胞,这为体外筛选安氏隐孢子虫免疫相关蛋白基因提供了理论依据。为筛选安氏隐孢子虫免疫相关蛋白基因和新的疫苗候选抗原基因,本研究构建了安氏隐孢子虫T7噬菌体展示文库,用犊牛小肠上皮细胞对文库进行筛选,获得了部分安氏隐孢子虫免疫相关蛋白基因,将获得的基因分别在原核系统和真核系统中表达后,免疫小鼠进行体液免疫和细胞免疫水平的检测,然后对小鼠进行微小隐孢子虫攻虫实验,从而验证重组抗原亚单位疫苗及核酸疫苗的交叉保护性。结果显示成功构建了安氏隐孢子虫T7噬菌体展示文库,文库容量为1.2×108 pfu/mL,重组率为90%,扩增后文库滴度为2.4×1010 pfu/mL。经过3次每次5轮的筛选,获得了17个新的安氏隐孢子虫免疫相关蛋白基因,将其中的两个基因命名为C.andersoni 2(CA2)和C.andersoni 42 (CA42),通过NCBI比对分析显示,CA2为安氏隐孢子虫未知功能蛋白,CA42为安氏隐孢子虫肌动蛋白。将CA2与CA42扩增后,构建了pET-28a-CA2和pET-28a-CA42重组原核表达质粒;将重组原核表达质粒分别转化到感受态大肠埃希菌DE3内,通过IPTG进行诱导表达,Western Blotting显示重组蛋白大小约为15ku和19ku,均能被安氏隐孢子虫免疫小鼠的多克隆抗体识别。将重组蛋白免疫BALB/c小鼠后,体液免疫水平ELISA检测结果显示,试验组血清中抗体滴度随免疫次数增加而逐渐升高,第三次免疫后抗体滴度与对照组相比差异均显著(P<0.05)。细胞免疫水平检测显示,与对照组相比,CD4+ T淋巴细胞数差异均显著,CD8+ T淋巴细胞数与佐剂对照组和空白对照组相比差异均不显著(P>0.05),CD4+/CD8+ T淋巴细胞数的值与佐剂对照组和空白对照组相比差异均显著(P<0.05)。免疫后,对小鼠分别接种1×106个微小隐孢子虫卵囊,通过克粪便卵囊数的检查,结果显示,从第三天起各实验组克粪便卵囊数有所下降。CA2组减虫率为37.5%,而CA42组减虫率为32.2%,与对照组相比差异均显著(P<0.05)。结果表明重组蛋白具有一定的免疫原性和交叉保护性。将CA2与CA42基因分别定向亚克隆到真核表达质粒pVAX1中,构建pVAX1-CA2和pVAX1-CA42重组真核表达质粒。将重组质粒成功转染HeLa细胞后,提取细胞蛋白,通过Western Blotting显示重组蛋白大小与预期相符且均能被安氏隐孢子虫免疫小鼠的多克隆抗体识别。将重组质粒免疫BALB/c小鼠,同对照组相比,体液免疫水平差异均显著(P<0.05),从免疫后第2周开始pVAX1-CA2免疫组抗体水平高于pVAX1-CA42免疫组。CD4+ T细胞亚类数量显著高于对照组(P<0.05),CD4+/CD8+ T细胞亚类数量显著高于pVAX1对照组(P<0.05)。交叉保护性实验结果显示, CA2组减虫率为34.6%,而CA42组减虫率为25.8%,与对照组相比差异均显著(P<0.05)。结果表明重组核酸疫苗对微小隐孢子虫具有一定的交叉保护性。本研究为隐孢子虫病的防治提供了一定的理论基础和全新的研究方向。更多还原

【Abstract】 Cryptosporidium is an important parasite which parasite on the respiratory and digestive tract epithelial cells of human and spinal animals. Cryptosporidiosis is caused by it in the clinical manifestations of severe watery diarrhea disease. In 1907, Tyzzer first reported the case that mice infected with Cryptosporidium. Until 1982, the U.S. Centers for Disease Control reported that the 21 male AIDS patients in six U.S. cities caused the severe diarrhea with cryptosporidiosis. Cryptosporidium was researched over the world from then on. Cryptosporidiosis has been listed as one of the United States six diarrheal disease. In 2003, Cryptosporidiosis was included in the " prevention and application of emerging infectious diseases" that was major scientific and technological projects by China’s Ministry of Science, and was one of two parasitic diseases. Cryptosporidium species have been discovered more than 20 strains so far, but most scientists believe that only 19 species are valid species. Cryptosporidium andersoni is one of them. In 2000, it was named as Cryptosporidium andersoni. The adult cattle was mainly infected with Cryptosporidium andersoni. The cattle infected with C. andersoni can cause milk production decreased, weight loss, and it is affected the development of dairy industry. In recent years, C. andersoni was identified by Leoni and Morse in the human body. Although cryptosporidiosis has been studied for many years at home and abroad, but there is no effective preventive measures and drugs. More than 200 kinds of drugs were selected, including all of the antibiotics, sulfa drugs and anticoccidial drugs for the disease, but no drug has effect for Cryptosporidium. So immunization is the main way to control the disease, and the key is to screen effective anti-Cryptosporidium vaccine candidate antigens.Cryptosporidium andersoni is a parasitic protozoa. There was report that it infected human. At present, the reports of C. andersoni has focused on epidemiological surveys. There are no report about Cryptosporidium andersoni vaccine candidated gene screening and cross-protection studies. Cryptosporidiosis is related with the host immune system. So antibodies and vaccines will play an important role for treatment and prevention of the disease. Received Cryptosporidium andersoni vaccine candidate genes and cross-protection study will provide a new research directions of the prevention and treatment of cryptosporidiosis. C. andersoni vaccine candidate genes is important problems to be solved. The method of screening immune-related genes through immune protein gene library is a common technical methods. The phage display technology is an new biotechnology of antigen gene screened. This technique has been widely used in the field of life science research. There is no reported that T7 phage display library screening is used for screening immune-related genes in C. andersoni. In 2003, Andre successfully proved that Human and bovine epithelial cells were infected by Cryptosporidium andersoni in vitro. It provides a theoretical basis for screening of Cryptosporidium andersoni immune-related protein gene in vitro.To screen of C. andersoni immune protein gene and the new vaccine candidate antigen genes, T7 phage display library of Cryptosporidium andersoni has been constructed in this study. Immune-related protein gene was screened from the library using calf intestinal epithelial cells. two genes from the library were used in prokaryotic and eukaryotic expression system. Then the mice were immuned by the recombinant proteins. The level of humoral and cellular immune was detected and then the mice attacked Cryptosporidium parvum to verify the recombinant subunit vaccine and DNA vaccine cross-protection.The results showed that Cryptosporidium andersoni T7 phage display library was successful constructed. The titer of the amplified library was 1.2×108 pfu / mL. The library capacity was 2.4×1010 recons. PCR identification on 50 randomly selected plaques was conducted, the library recombination rate was 90%. The 80% insert segments were greater than 0.3kb. The amplified library was screened and at the fifth round of screening. 17 new immune-related protein gene of Cryptosporidium andersoni were identified. two of these genes named C.andersoni 2 (CA2) and 42 (CA42). They were analysised in NCBI. CA2 was Cryptosporidium andersoni unknown proteins, CA42 was Cryptosporidium andersoni actin.After the amplification of CA2 and CA42, recombinant prokaryotic expression plasmid pET-28a-CA2 and pET28a-CA42 were constructed. The recombinant prokaryotic expression plasmids were transformed into competent E. coli DE3. The expression was induced by IPTG. Western Blotting show that the size of the recombinant protein are about 15ku and 19ku and the protein can be identified by polyclonal antibody from immunized mice of C. andersoni. Then the BALB/c mice were immunized by recombinant protein. ELISA test showed the immune serum antibody titer of experimental group was gradually increased. After the third immunization, antibody titers show significantly increase compared with the control group (P<0.05) . Detection of cellular immunity showed that CD4+ T lymphocytes were significantly increase compared with the control group (P<0.05) and CD8+ T lymphocytes were not significantly increase (P>0.05). CD4+/ CD8+ T lymphocytes were significantly increase compared with the control group (P<0.05). After immunization, mice were inoculated with 1×106 Cryptosporidium parvum oocysts. The results show that the oocysts number of grams in the experimental group fecal were decreased from the third day of inoculation. Compared with the control group. CA2 group reduction rate was 37.5% and CA42 Group 32.2%. There were significantly increased (P<0.05).The results showed that the recombinant protein has immunogenicity and cross-protection.The CA2 and CA42 genes were subcloned into the eukaryotic expression plasmid pVAX1. Recombinant eukaryotic expression plasmid pVAX1-CA2 and pVAX1-CA42 were constructed. The recombinant plasmid was successfully transfected into HeLa cells, then extracted cell protein. Western Blotting show that the recombinant protein was conformed with expectation and was recognized by polyclonal antibody of immunized mice of C.andersoni. Then BALB/c mice were immuned by recombinant plasmid. Compared with the control group, the level of humoral immunity was significantly increas (P<0.05), CD4+ T cells was higher than control group (P<0.05). CD4+/CD8+ T cells were higher than pVAX1 group (P<0.05). Cross-protection experiments prove that CA2 group reduction rate was 34.6%, while CA42 group was 25.8%. Compared with the control group, there were significantly increased (P <0.05). The results show that recombinant DNA vaccine has some cross-protection against Cryptosporidium parvum. This study will be a theoretical basis of the prevention and treatment of cryptosporidiosis andersoni and cross-protective vaccine research.更多还原