隐孢子虫鼠基因型三个核酸疫苗、亚单位疫苗的制备及其免疫保护性研究

发布时间：2018-03-19 00:03

本文选题：隐孢子虫鼠基因型　切入点：核酸疫苗　出处：《中国农业科学院》2010年硕士论文　论文类型：学位论文

【摘要】： 隐孢子虫病(cryptosporidiosis)是由隐孢子虫(Cryptosporidium spp.)引起的一种以持续性腹泻为主要临床特征的全球性人兽共患寄生虫病。隐孢子虫病具有广泛的宿主类型,其病原可以寄生于哺乳类、鸟类、爬行类、两栖类、鱼类等包括人类在内的170多种动物。隐孢子虫病给畜牧业生产造成严重的经济损失,威胁免疫功能缺陷者的生命安全。当前,对隐孢子虫病的研究已经成为全球寄生虫学领域的热点之一。目前还没有治疗隐孢子虫病的特效药物,因此隐孢子虫病的免疫预防研究就显得愈发重要。本研究选取了隐孢子虫鼠基因型子孢子表面蛋白P23、CP15/60以及卵囊壁蛋白CP41 3个主要保护性抗原基因,利用DNA重组技术将这3个基因分别克隆到真核表达载体pVAX1上,制成核酸疫苗。用制备的3个隐孢子虫核酸疫苗免疫BALB/c小鼠,对疫苗诱导的IgG水平、小鼠排卵囊量情况进行了检测。结果显示,免疫小鼠血清中特异性抗体IgG滴度水平随免疫次数的增加而增加,呈现逐步升高的趋势,在第3次免疫后达到最高。实验组IgG滴度水平极显著高于对照组(P0.01),而实验组内、对照组组内差异不显著(P0.05)。与生理盐水、pVAX1对照组相比,实验组小鼠卵囊排出数量显著小于对照组(P0.05),减卵率在85% ~100%之间,而实验组内、对照组组内差异不显著(P0.05);试验组小鼠开始排出卵囊时间延后,排出卵囊的持续时间缩短。表明本研究制备的3个核酸疫苗可以诱导小鼠产生良好的特异性免疫应答,对BALB/c小鼠隐孢子虫鼠基因型感染有较好的免疫保护作用。将重组质粒pGEX-4T-1-P23、pGEX-5x-3-CP41、pET-28b(+)-CP15/60在大肠杆菌中成功表达。Western blot分析表明制备的3个重组蛋白具有良好的免疫原性,可被隐孢子虫鼠基因型感染小鼠血清所识别。将3个重组蛋白与弗氏佐剂充分乳化后制成亚单位疫苗免疫BALB/c小鼠,对疫苗诱导的IgG水平、小鼠排卵囊情况进行了检测。间接ELISA检测结果表明重组蛋白rP23、rCP41、rCP15/60可引起小鼠产生良好的体液免疫应答,免疫小鼠血清中特异性抗体IgG滴度水平随免疫次数的增加而增加,呈现逐步升高的趋势,在第3次免疫后达到最高,实验组IgG滴度水平极显著高于对照组(P0.01)。与PBS、佐剂、rGST(4T)和rGST(5x)对照组相比,实验组卵囊排出量显著低于对照组(P0.05),减卵率在73%~80%之间,而实验组内、对照组组内差异不显著(P0.05);试验组开始排出卵囊时间延后,排出卵囊持续时间缩短。表明本实验制备的3个亚单位疫苗可明显地诱导小鼠产生特异性免疫应答,对BALB/c小鼠隐孢子虫鼠基因型感染有较好的免疫保护作用。本实验对隐孢子虫病的免疫预防进行了初步的探索,制备的3个核酸疫苗和3个亚单位疫苗对隐孢子虫病的疫苗研究进行了有益的补充,为下一步深入研制具有高保护性的隐孢子虫预防疫苗提供基础。
[Abstract]:Cryptosporidiosism is a global zoonosis caused by Cryptosporidium spp.which is characterized by persistent diarrhea. Cryptosporidiosis has a wide range of host types and can parasitize mammals and birds. Reptiles, amphibians, fish, etc. More than 170 species of animals, including human beings. Cryptosporidiosis causes serious economic losses to animal husbandry and threatens the lives of people with immune deficiency. The research on Cryptosporidiosis has become one of the hotspots in the field of parasitology. There are no effective drugs to treat Cryptosporidiosis, so the immunological prevention of Cryptosporidiosis becomes more and more important. In this study, three major protective antigen genes of Cryptosporidium genetype P23 CP15 / 60 and oocystin wall protein CP41 were selected and cloned into eukaryotic expression vector pVAX1 by DNA recombination technique. Nucleic acid vaccine was prepared. BALB/c mice were immunized with three Cryptosporidium nucleic acid vaccines. The levels of IgG induced by the vaccine and the number of ovulation sac of the mice were determined. The titer of specific antibody IgG in serum of immunized mice increased gradually with the increase of immunization times, and reached the highest level after the third immunization. The titer of IgG in the experimental group was significantly higher than that in the control group (P 0.01), while in the experimental group, the titer of IgG was significantly higher than that of the control group (P 0.01). Compared with normal saline pVAX1 control group, the number of oocysts in the experimental group was significantly lower than that in the control group (P 0.05), and the oocyte reduction rate was between 85% and 100% in the control group, while in the experimental group, the number of oocysts in the control group was significantly lower than that in the control group. In the control group, the difference was not significant (P0.05), the time of oocyst excretion was delayed and the duration of oocyst excretion was shortened in the experimental group. The results showed that the three nucleic acid vaccines prepared in this study could induce the mice to produce a good specific immune response. It can protect the BALB/c mice against Cryptosporidium genetype infection. The recombinant plasmid pGEX-4T-1-P23 pGEX-5x-3-CP41 pET-28b (pET-28b) was successfully expressed in Escherichia coli. Western blot analysis showed that the three recombinant proteins had good immunogenicity. Three recombinant proteins were emulsified with Freund's adjuvant and subunit vaccine was prepared to immunize BALB/c mice. The results of indirect ELISA test showed that the recombinant protein rP23rCP41 rCP15 / 60 could induce a good humoral immune response in mice, and the titer of specific antibody IgG in serum of immunized mice increased with the increase of immunization times. The level of IgG titer in the experimental group was significantly higher than that in the control group (P 0.01). Compared with the PBSs, adjuvant rGST4T) and rGST5x), the egg sac excretion in the experimental group was significantly lower than that in the control group (P 0.05), and the egg reduction rate was between 7380% and 73.8%. In the experimental group, there was no significant difference between the control group and the control group (P 0.05), but the time of oocyst excretion in the experimental group was delayed and the duration of the oocyst excretion was shortened. The results showed that the three subunit vaccines prepared in this experiment could induce the specific immune response of the mice. It can protect the BALB/c mice against Cryptosporidium genetype infection. Three nucleic acid vaccines and three subunit vaccines were prepared to supplement the research of Cryptosporidiosis vaccine. It provides the foundation for the further development of the highly protective Cryptosporidium prophylaxis vaccine.
【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】：2010
【分类号】：R392

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