微小隐孢子虫Rhomboid蛋白抗原定位与免疫保护性研究

发布时间：2018-10-25 08:42

【摘要】： 隐孢子虫(Cryptosporidium)是一种寄生性原虫,主要寄生于人和其他哺乳动物的胃肠道、禽类的呼吸道等粘膜上皮细胞内,引起隐孢子虫病。该病分布广泛,人畜感染率高,且危害严重,临床上以严重水样腹泻为主要特征。目前已有200余种药物被试用于治疗该病,但尚无特效药物。有学者通过对隐孢子虫亚单位疫苗及核酸疫苗的研究认为有效的疫苗对该病的防治具有一定的作用,关键在于选择有效的疫苗候选抗原。Rhomboid蛋白是顶器门原虫侵入与粘附宿主细胞的重要蛋白,有望成为疫苗的候选抗原。为探讨微小隐孢子虫Rhomboid基因对微小隐孢子虫感染的免疫保护力,本试验利用生物信息学技术从微小隐孢子虫基因组cDNA中预测Rhomboid基因开放阅读框,设计引物,从微小隐孢子虫长春株总RNA中扩增出Rhomboid基因,将其克隆入原核表达载体并表达出微小隐孢子虫重组Rhomboid蛋白,利用间接免疫荧光技术对该蛋白进行抗原定位;同时用DNA重组技术将微小隐孢子虫Rhomboid基因克隆入真核表达载体构建核酸疫苗,将所构建的微小隐孢子虫Rhomboid重组蛋白与核酸疫苗分别免疫小鼠检测其免疫保护力。结果显示:扩增出1395bp的开放阅读框,经Blast比对显示核苷酸和氨基酸的同源性分别为99.9%和99.6%。并表达出51KDa的重组融合蛋白,Western blot试验证实所得蛋白具有反应原性,间接免疫荧光显示该蛋白位于子孢子;用间接免疫荧光方法在重组质粒转染后的Hela细胞中检测到特异蛋白,通过Western blot试验证实所得蛋白具有反应原性;重组Rhomboid蛋白免疫保护性试验结果显示,随着免疫次数的增加抗体滴度逐渐增加,试验组与对照组相比差异极显著(P0.01)。CD4+、CD8+T淋巴细胞数与对照组相比差异均不显著(P0.05)。试验组小鼠对抗微小隐孢子虫感染具有一定保护作用,卵囊排出量明显减少,排出时间缩短2d,减虫率达65.48%。核酸疫苗免疫保护性试验结果表明,试验组与对照组相比,特异性抗体滴度显著增加(P0.01);CD4+、CD8+T淋巴细胞数与对照组相比差异均不显著(P0.05)。攻虫后试验组小鼠卵囊排出量明显减少,排出时间缩短2d,减虫率达63.1%。
[Abstract]:Cryptosporidium (Cryptosporidium) is a parasitic protozoa, mainly parasitic in human and other mammalian gastrointestinal tract, poultry respiratory tract and other mucosal epithelial cells, causing Cryptosporidiosis. The disease is widely distributed, the infection rate of human and animal is high, and the harm is serious. The main clinical characteristic of the disease is severe watery diarrhea. At present, more than 200 drugs have been tested for the treatment of the disease, but there are no special drugs. Through the research of Cryptosporidium subunit vaccine and nucleic acid vaccine, some scholars think that the effective vaccine has certain effect on the prevention and cure of the disease. The key lies in the selection of effective vaccine candidate antigens. Rhomboid protein is an important protein which invades and adheres to host cells and is expected to be a candidate antigen for vaccine. In order to study the immune protection of Cryptosporidium microsporidium Rhomboid gene against Cryptosporidium minimus infection, the open reading frame of Rhomboid gene was predicted from genomic cDNA of Cryptosporidium minimus by bioinformatics technique, and primers were designed. The Rhomboid gene was amplified from the total RNA of Cryptosporidium minimus Changchun strain and cloned into prokaryotic expression vector to express the recombinant Rhomboid protein of Cryptosporidium minimus. At the same time, Rhomboid gene of Cryptosporidium minimus was cloned into eukaryotic expression vector by DNA recombination technique to construct nucleic acid vaccine. The recombinant Rhomboid protein of Cryptosporidium minimus and the nucleic acid vaccine were immunized to test the immune protection of mice. The results showed that the open reading frame of 1395bp was amplified and the homology of nucleotide and amino acid was 99.9% and 99.66% respectively by Blast comparison. The recombinant fusion protein of 51KDa was confirmed to be reactive by, Western blot assay, and the specific protein was detected by indirect immunofluorescence in Hela cells transfected with recombinant plasmid. The results of Western blot test showed that the antibody titer of recombinant Rhomboid protein increased with the increase of immunization times. Compared with the control group, the difference was very significant (P0.01). There was no significant difference in the number of CD4, CD8 T lymphocytes between the experimental group and the control group (P0.05). The mice in the test group had a protective effect against Cryptosporidium minimus infection, the oocyst excretion was obviously reduced, the excretion time was shortened by 2 days, and the worm abatement rate was 65.48. The results of immune protection test showed that the titer of specific antibody in the test group was significantly higher than that in the control group (P0.01), while the number of CD4 and CD8 T lymphocytes was not significantly different from that of the control group (P0.05). The oocyst excretion of the test group was obviously reduced, the excretion time was shortened by 2 days, and the worm reduction rate was 63.1%.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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