【作者】 肖丹；

【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】 为获得微小隐孢子虫表面粘附相关蛋白基因,从而获得疫苗的候选基因。本研究构建微小隐孢子虫核糖体展示文库,利用新生犊牛小肠上皮细胞对体外翻译蛋白质进行黏附筛选。经筛选得到的基因用ScanProsite和InterProScan分析推测氨基酸的序列。构建针对新基因的原核表达载体,获得重组蛋白。将重组蛋白免疫BALB/c小鼠,利用间接荧光抗体技术对重组蛋白进行微小隐孢子虫卵囊或子孢子抗原定位,同时进行重组蛋白对BALB/c小鼠的免疫保护性试验。另构建重组真核表达质粒,将其转染Hela细胞后,经G418加压筛选获得亚克隆细胞株。用间接免疫荧光抗体及Westen-blot方法检测重组真核表达质粒是否能在真核细胞中表达相应的目的蛋白及其反应原性。将真核重组质粒滴鼻注射免疫BALB/c小鼠及怀孕山羊(3-4个月),检测真核重组质粒的免疫保护性。试验结果表明:成功合成隐孢子虫双链cDNA,容量为1.3×10~ 13个DNA分子,在核糖体展示系统进行体外翻译。经筛选获得新的6个微小隐孢子虫粘附相关蛋白基因。选择开放阅读框长度为585bp,编码粘蛋白样的糖蛋白的基因作为疫苗的候选基因(命名为CP585)进行免疫保护性研究。本研究成功构建pET-28a-CP585原核表达载体,获得CP585的重组蛋白。经间接免疫荧光抗体试验证实CP585蛋白位于微小隐孢子虫卵囊壁表面和子孢子表膜。将纯化的重组蛋白免疫BALB/小鼠,诱导机体产生了特异的体液和细胞免疫应答。用1×10~6个微小隐孢子虫卵囊对小鼠进行攻虫感染,试验组与各对照组相比排出卵囊明显减少(P < 0.05),减虫率为47.78%。构建的真核重组质粒pVAX1-CP585,经检测可以在体外表达特异性蛋白质。用该重组质粒滴鼻免疫BALB/c小鼠,证实可诱导BALB/c小鼠产生体液免疫和细胞免疫应答,并能对微小隐孢子虫的感染有一定抵抗作用,减虫率达60.52%。用该重组质粒滴鼻免疫怀孕山羊(3-4个月),经检测山羊血清中有抗微小隐孢子虫特异性抗体,且CD4+/ CD8+比值升高。山羊的子代羔羊出生后人工灌胃感染1×10~6微小隐孢子虫卵囊,经检测pVAX1-CP585试验组比各对照组相卵囊排出量明显减少(P<0.05),减虫率为49.29%。说明免疫真核重组质粒山羊的母体中含有抗微小隐孢子虫特异性抗体,对其易感子代有一定的免疫保护力。本研究证实CP585基因可以作为微小隐孢子虫疫苗的候选基因。更多还原

【Abstract】 Cryptosporidium parvum ribosome display library was screened with intestinal epithelial cells（IECs）according to established method in our laboratory. Subsequently, Protein was selectively and specifically bound to IECs using a multi-step panning procedure.Amino acid sequence was analysised by ScanProsite and InterProScan. The target gene was cloned into Pet-28a and expessed in BL21. The expressive levels were determined by SDS-PAGE and Western -blot analysis of the cell lysates.The localization of the protein（the expressive product of the target gene）was determined by immunofluorescent-antibody technique using specific antibodies. The BALB/c mice were immunized with the recombiant protein to detect the responses of specific humoral and cell immunity. The efficacy of recombiant protein was investigated in BALB/c mice.At the same time, the target gene was inserted into eukaryotic expression vector pVAX1 and transfected into Hela cell. The expression cell strain was selected with G418. The specific reombiant proteins were detected in Hela cells by indirect Immune fluorescence and Westen blot assay. The BALB/c mice and the adult pregnat goats were immunized with the reombiant eukaryotic expression vector to detect the responses of specific humoral and cell immunity. And then its efficacy was investigated in BALB/c mice and the adult pregnat goats.The results were as follows:Constrution of the ribosome display library of C.parvum.There were 1.3×10 ¹³DNA in this library and translated on ribsome using the kit （Promega） to generateternary PRM complexes. After five rounds of selection,six genes of C. parvum were selected. The gene which has an N-terminal signal peptide and transmembrane regions was named CP585.Construction and expression of prokaryotic expression vector.The recombinant plasmids pET-28a-CP585 was constructed and expressed in E.coli host cells BL21.The recombinant proteins were purified and detected its specificity by Western-blot. The mice were immunized with CP585 protein that indicated the responses of specific antibodies and cell immunity.The result of immune protect experiment showed that oocysts number of immunized mice was decreased significantly compared with control groups. The decreased worn rate of the experiment was 47.87%. Immunofluorenscence staining showed that CP585 protein was present on the surface of sporozoites and oocysts.Construction and expression of the Eukaryotic expression vector. The recombinant plasmids pVAX1-CP585 was constructed by cloning the CP585 gene into eukaryotie expression vecto pVAX1 and expressed in Hela cells.After transfected intoHela cells,the expression cell strains were selected with G418. The specific recombiant protein was detected in Hela cell strains by indirect immunofluorescence assay and Western-blot. The BALB/c mice were immunized with pVAX1-CP585 plasmid.The responses of pecific humoral and cell immunity was significant （ P<0.05）. The experiment that the BALB/c mice were inculated with C.parvum oocysts showed pVAX1-CP585 had immuno-protective effect on C.parvum infection, the decreased worn rate of the experiment was 60.52%. The adult pregnat goats were inoculated intranasally with pVAX1-CP585 pasmid. The results showed that pVAX1-CP585 could induce immune response of goats and the vaccinated goats can tranfer the immunity to offspring conferring protection against C.parvum infection.Oocyst number shedding by the kids of experiment were shorter than those of control groups.The decreased worn rate of the experiment was 49.29%.The study laid a good foundation of the further research about prevention and cure of cryptosporidiosis.更多还原