微小隐孢子虫粘附相关蛋白基因的筛选及免疫特性研究

发布时间：2018-07-14 19:27

【摘要】： 隐孢子虫(Cryptosporidium)是一种引起人和动物腹泻的重要的寄生性原虫,由其引起的隐孢子虫病(Cryptosporidiosis)是重要的公共卫生问题。目前尚无理想预防隐孢子虫病的疫苗。本研究采用新生犊牛小肠上皮细胞对本室构建的微小隐孢子虫cDNA核糖体展示文库进行筛选,获得新的微小隐孢子虫粘附相关蛋白基因,并用分子生物学方法对其进行了原核表达,进而免疫BALB/c小鼠研究其免疫保护力,通过免疫荧光技术对重组蛋白进行定位。应用DNA重组技术将该基因克隆入真核表达载体,并将重组质粒转染Hela细胞,验证其在真核细胞中的表达,同时进行动物保护性试验,观察重组质粒的免疫保护力。结果成功的筛选到一个新的微小隐孢子虫粘附蛋白相关基因,并将其命名为CP16。测序结果显示CP16长度为525bp,含有一大小为423bp的开放阅读框,由ScanProsite与InterProScan分析推测的氨基酸序列,71-93氨基酸是跨膜区包含一个跨膜区,一个N端糖基化位点。构建了微小隐孢子虫粘附相关蛋白基因CP16的原核表达质粒pET-28a-CP16,并转化Rosetta(DE3)后获得了表达,表达产物以包涵体形式存在。免疫荧光检测证实CP16蛋白位于子孢子表膜和卵囊壁表面。动物试验结果显示重组CP16蛋白具有一定的保护力,减虫率可达51.17%。构建的真核表达质粒在Hela细胞中表达成功;应用pVAX1-CP16免疫BALB/c小鼠,结果表明可诱导产生体液免疫和细胞免疫应答。试验组小鼠血清中IgG滴度随免疫次数增加而逐渐升高,三免后实验组IgG滴度与对照组相比差异极显著(P0.01)。试验组小鼠的CD4+ T淋巴细胞数和CD4+/CD8+ T淋巴细胞数的值与对照组相比差异显著(P0.05)。小鼠免疫后经口接种微小隐孢子虫卵囊,试验组与对照组相比卵囊排出量明显减少,免疫组减虫率可达到58.68%。将pVAX1-CP16经鼻粘膜接种妊娠山羊,并对免疫山羊的后代进行攻虫试验。结果表明重组质粒能够诱导母羊产生免疫反应,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显著(p0.05)。羔羊免疫后经口接种微小隐孢子虫卵囊,与对照组相比试验组羔羊每天排出卵囊数量明显减少,排卵囊持续时间较短,减虫率达到56.1%。
[Abstract]:Cryptosporidium (Cryptosporidium) is an important parasitic protozoa causing human and animal diarrhea. The Cryptosporidium disease (Cryptosporidiosis) caused by the Cryptosporidium is an important public health problem. There is no ideal vaccine to prevent Cryptosporidium. This study uses the small cryptospore constructed by the small intestinal epithelial cells of newborn calves. The cDNA ribosome display library was screened to obtain a new gene for the adhesion related protein of Cryptosporidium parvum, and its prokaryotic expression was expressed by molecular biological methods. Then the immune protective ability of BALB/c mice was studied. The recombinant protein was located by immunofluorescence technology, and the gene was cloned by DNA recombination technology. The recombinant plasmid was transfected into Hela cells to verify its expression in the eukaryotic cells. At the same time, the protective test of the recombinant plasmid was carried out and the immune protection of the recombinant plasmid was observed. A new gene of the adhesion protein of Wei Xiaoyin Sporozoa was successfully screened and named as CP16. sequencing results, the length of CP16 was 525bp, An open reading frame with a size of 423bp was used to analyze the sequence of amino acids conjectured by ScanProsite and InterProScan. The 71-93 amino acid was a cross membrane region containing a transmembrane region and a N end glycosylation site. The prokaryotic expression of pET-28a-CP16 of the expression of CP16 of the adhesion related protein gene of Cryptosporidium was constructed, and Rosetta (DE3) was converted to Rosetta (DE3). The expression products were expressed in inclusion body form. Immunofluorescence detection showed that the CP16 protein was located on the surface of the sporozoite membrane and the oocyst wall. The experimental results showed that the recombinant CP16 protein had certain protective ability. The eukaryotic expression plasmid constructed by the worm reduction rate of 51.17%. was successful in the Hela cells; pVAX1-CP16 immunized with BALB/c mice. The results showed that the humoral and cellular immune responses were induced. The IgG titer in the test group increased with the increase of the number of immune times. The IgG titer of the experimental group was significantly different from that of the control group (P0.01). The value of the number of CD4+ T lymphocytes and the number of CD4+/CD8+ T lymphocytes in the experimental group was significantly different from that of the control group. (P0.05). The mice immunized with the oocyst of Cryptosporidium parvum after immunization, compared with the control group, the amount of oocyst excretion decreased significantly compared with the control group. The rate of worm reduction in the immune group could reach 58.68%. by inoculating pVAX1-CP16 through the nasal mucosa and carrying out the attack test on the offspring of the immune goats. The results showed that the recombinant plasmid could induce the immune reaction of the ewes. As the number of immunization increased gradually, the test group had a significant difference compared with the control group (P0.05). The lambs were inoculated with the oocyst of Cryptosporidium parvum after immunization. Compared with the control group, the number of oocyst excreted per day in the experimental group decreased significantly, the duration of ovulation sac was shorter and the rate of worm reduction reached 56.1%..
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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