【作者】 郑君；

【导师】 张西臣；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 博士

【摘要】 随着现代化养鸡业的发展,鸡球虫病已成为四季多发性疾病,预防和治疗球虫病目前已成为研究的焦点。由于长期以来传统药物的使用,使得球虫产生一定的耐药性,再加上药物残留等问题使得球虫病的防治愈发困难。免疫预防是控制该病的主要手段,但是因为球虫生活史复杂,侵入机制尚不清楚,目前行之有效的疫苗较少,对于疫苗候选基因的选择亟待解决。微线蛋白作为一种粘附相关蛋白,由位于虫体顶端的微线体分泌,是虫体识别和结合宿主细胞受体的关键性蛋白。Rhomboid蛋白自发现以来一直吸引着研究者的注意。作为一类膜内的丝氨酸蛋白酶,它参与着表皮生长因子及表皮生长因子受体信号的转导。近年来人们在很多顶器门的原虫体内也发现了Rhomboid蛋白的存在,其中包括柔嫩艾美耳球虫。经过对弓形虫和疟原虫Rhomboid蛋白的鉴定,发现Rhomboid蛋白水解其底物MIC蛋白是虫体侵入宿主细胞的关键过程,而柔嫩艾美耳球虫Rhomboid蛋白的功能还未见报道,它能否水解MIC蛋白,其水解过程是不是球虫侵入的关键过程还有待研究。酵母作为一种提高免疫力的佐剂,早已经被人们研究和关注。酵母细胞中含有大量的葡聚糖和甘露寡糖,可以提高免疫能力,增加抗体效价和外周血中淋巴细胞的数量,从而增加机体的抗菌抗癌能力。然而酵母作为免疫佐剂的应用方向还有待进一步拓宽,在防治寄生虫病方面将具有意义深远的研究价值。3,4二氯异香豆素(DCI)是一种非常有效的丝氨酸蛋白酶抑制剂,它能够使大多数丝氨酸蛋白酶失去活性,据报道,DCI可以抑制果蝇属Rhomboid-1的酶活性,影响其EGFR的信号转导；也能够抑制弓形虫TgROM5的活性,影响速殖体侵入宿主细胞。而DCI是否能够抑制柔嫩艾美耳球虫Rhomboid的活性,从而影响其侵入,目前还未见报道。本研究首先根据CytoTrap酵母双杂交系统,验证了E. tenella Rhomboid蛋白对温度敏感型酵母菌株cdc25H存在自激活作用；然后根据Ga14酵母双杂交系统,成功构建诱饵表达质粒pGBKT7-Rho与捕获质粒pGADT7-MIC1和pGADT7-MIC4,通过诱饵质粒与捕获质粒共转化酵母细胞AH109,从而筛选出与Rhomboid蛋白相互作用的MIC4蛋白；免疫共沉淀进一步验证Rhomboid与MIC4蛋白存在相互作用：将含有互作蛋白的酵母细胞AH109破碎后免疫雏鸡,攻虫试验证明互作蛋白对雏鸡具有一定的免疫保护作用,抗球虫指数可以达到168.24；最后验证了DCI体外对子孢子侵入细胞的抑制作用。为柔嫩艾美耳球虫侵入细胞的分子机制研究奠定了基础,也为球虫病的防治提供了理论依据。E.tenella Rhomboid蛋白CytoTrap酵母双杂交系统自激活作用的鉴定通过RT-PCR扩增Rhomboid基因并对其分析,然后根据CytoTrap酵母双杂交系统构建诱饵质粒pSos-Rho,将诱饵质粒与对照质粒共转化酵母细胞中,从而验证Rhomboid蛋白的自激活作用。结果表明,成功扩增了柔嫩艾美耳球虫Rhomboid基因开放阅读框并构建了重组诱饵表达载体pSos-Rho,同源性比对分析得出Rhomboid蛋白即柔嫩艾美耳球虫EtROM3.Western blot显示诱饵蛋白在酵母细胞中可以成功表达,间接免疫荧光将重组蛋白定位于宿主酵母cdc25H细胞膜附近。通过将诱饵质粒与对照质粒共转化酵母细胞中,结果显示重组诱饵融合蛋白激活了宿主菌cdc25H的Ras途径,导致酵母细胞在37℃半乳糖培养基上能够生长,说明诱饵质粒对于CytoTrap酵母双杂交系统存在自激活作用,从而不能使用本系统进行互作蛋白的筛选。E.tenella Rhomboid蛋白相互作用的MIC蛋白的筛选首先通过RT-PCR扩增柔嫩艾美耳球虫MIC1和MIC4基因片段,然后根据Ga14酵母双杂交系统构建诱饵质粒pGBKT7-ROM,捕获质粒pGADT7-MIC1和pGADT7-MIC4,将捕获质粒分别与诱饵质粒共转化酵母细胞AH109中,验证蛋白的交互作用。结果表明,重组诱饵质粒与捕获质粒均构建成功,目的基因可以在感受态酵母细胞中表达,诱饵蛋白对酵母细胞既没有毒性,也不存在自激活作用。通过Ga14酵母双杂交系统验证,Rhomboid蛋白与MIC4蛋白存在相互作用,Western blot鉴定结果显示MIC4蛋白长度变小,推测是由于Rhomboid蛋白酶的切割作用所致。Rhomboid与MIC4蛋白相互作用的鉴定构建重组原核表达质粒pET28a-MIC4,重组真核表达质粒pcDNA-Myc-ROM和pcDNA-HA-MIC4,对柔嫩艾美耳球虫MIC4蛋白进行原核表达,纯化及复性,从而通过免疫共沉淀鉴定Rhomboid与MIC4蛋白间的互作；然后将构建的重组真核表达质粒共转染HeLa细胞,免疫共沉淀鉴定Rhomboid蛋白与MIC4蛋白间的相互作用。结果表明,重组原核与真核表达质粒均构建成功；将原核表达的Rhomboid与MIC4蛋白混合,通过免疫共沉淀说明,两种蛋白存在相互作用；将真核重组质粒共转染HeLa细胞,间接免疫荧光显示Rhomboid蛋白与MIC4可以成功表达；将共转染重组质粒的HeLa细胞破碎后,通过免疫共沉淀以及Western blot鉴定,Rhomboid蛋白与MIC4蛋白存在相互作用,而且Rhomboid蛋白具有酶切活性,可以切割其底物MIC4蛋白。这个试验的结果为更深入的研究球虫侵入宿主细胞的分子机制提供了理论依据。酵母中互作蛋白的动物保护性研究将含有Rhomboid蛋白、MIC4蛋白以及互作蛋白的酵母细胞AH109破碎后免疫雏鸡,从而进行攻虫试验,观察酵母细胞中具有互相作用的两种蛋白对柔嫩艾美耳球虫的动物保护性。结果说明,通过体液免疫水平和细胞免疫水平的检测,各实验组与对照组相比差异均显著(P＜0.05),其中含有两种互作蛋白的实验组差异极显著(P＜0.01)；攻虫试验结果显示其ACI可以达到168.24；同时试验结果首次表明酵母细胞也能够产生一定的免疫保护作用。本试验为鸡球虫病疫苗的研究提供了理论依据,也为酵母在寄生虫病方面的应用研究奠定了基础。DCI对子孢子侵染细胞的体外抑制试验进行柔嫩艾美耳球虫的体外原代鸡肾细胞培养,将DCI分成两组,一组提前作用于子孢子,一组与子孢子共同培养,然后通过对第一代裂殖体、第二代裂殖体以及子代卵囊的数量统计,观察不同浓度DCI体外对球虫子孢子侵染细胞的抑制作用。结果说明,20μM的DCI即可有效抑制子孢子体外对鸡肾细胞的侵入,同时能够控制球虫子代卵囊的产出量,对球虫病有潜在的治疗作用,有望成为抗球虫药物的候选成分,为球虫病的治疗研究奠定基础。本试验结果表明DCI通过抑制Rhomboid蛋白酶活性可以间接抑制球虫子孢子对宿主细胞的侵染,更进一步说明Rhomboid的酶切作用在虫体侵入过程中的重要地位,同时研究结果也为球虫病的治疗提供了理论依据。更多还原

【Abstract】 With the development of modern poultry industry, coccidiosis has become a season of multiple disease. Prevention and treatment of coccidiosis has become the focus of research. Due to long-term use of traditional drug, drug resistant has emerged in coccidian. It is more difficult to control coccidiosis combined with the problem of drug residues. Immunization is available means of controlling the disease, but because of complexed life cycle and uncleared intrusive mechanism of the coccidia, the current vaccine was less effective. Choice of candidate genes for coccidiosis vaccines must be resolved as soon as possible. Microneme protein, an adhesion protein, is secreted by the microneme at the top of the sporozoite. MIC is a key protein which is used to recognize and bind the host cell receptors. Rhomboid protein has been attracted the researchers note all the time since it was discovered. As a class of intramembrane serine proteases, Rhomboid participate in the epidermal growth factor and epidermal growth factor receptor signal transduction. In recent years, many rhomboid proteins had also been found in some apicomplexan protozoa, including Eimeria tenella. After identification of rhomboid in Toxoplasma and Plasmodium, the result indicated that the proteolysis of rhomboid and MIC was a key process of host cell invasion. There is no reporte about E. tenella rhomboid protein function now. Whether MIC protein is hydrolyzed by the rhomboid, Whether the hydrolysis process is the key to the process of coccidia invasion, which are need to study.As an immune promoter, Yeast had already been studied and attention. A large number of glucan and mannan oligosaccharides are contained in yeast cells. Yeast can improve immunity, increase in antibody titer and the number of peripheral blood lymphocytes, thereby increasing the body’s anti-bacterial and anti-cancer ability. However, as the immune enhancer, application of yeast need to be further expanded. Yeast will have far-reaching research value when it used to control parasitic disease. 3,4-dichloroisocoumarin (DCI) is a very effective serine protease inhibitor. Majority of serine protease sctivity can be losed by DCI. According to reports, Drosophila Rhomboid-1’s activity could be inhibited by DCI and the EGFR signal transduction was influenced. Activity of Toxoplasma gondii TgROM5 could also be impacted. So the host cells invasion was inhibited. But there is no report has been published on DCI used to inhibit Eimeria tenella Rhomboid and against coccidiosis.In this study, first, suitability of E. tenella rhomboid for CytoTrap yeast two-hybrid system was detected. Then according to the Gal4 yeast two-hybrid system, bait plasmid pGBKT7-Rho, Capture plasmids pGADT7-MIC1 and pGADT7-MIC4 was successfully constructed. Plasmids were cotransformed into yeast cells AH109, respectively. E.tenella MIC4 was screened for interacted with Rhomboid protein. Then the interaction was further validated by coimmunoprecipitation. The yeast host cells which contained interacted protein was broken to immune chickens. Experiment of attacking E. tenella showed that chicken had got protective immunity. Anticoccidial index could reach 168.24. Finally, inhibited effect of sporozoites cell invasion was detected by DCI. It is the basis for molecular mechanism research of E. tenella invasion. It is also theoretical evidence for prevention and treatment of coccidiosis.Sequence analysis and verification of E. tenella rhomboid bait plasmid suitability for CytoTrap yeast two-hybrid system The rhomboid gene segment of E. tenella was generated and analyzed by RT-PCR with primers. According to the CytoTrap yeast two-hybrid system, rhomboid gene was subcloned into the plasmid pSos. Then bait and control plasmids were cotransformed into yeast cells cdc25H. Self-activation of Rhomboid was verified. The results showed that E. tenella Rhomboid gene open reading frame was successfully amplified. Recombinant expression vector bait pSos-Rho was constructed. Compared with other amino acid sequence of rhomboid-like protein, we supposed that EtROMz is EtROM3 in E. tenella. Western blot showed that the bait protein in yeast cells could be successfully expressed. Rhomboid was located in the vicinity of the host cell membrane of yeast cdc25H by indirect immunofluorescence. When bait and control plasmids were cotransformed into cells, ras pathway was activated and yeast cells grew on galactose medium at 37 ℃. That mean bait protein could self-activation. Thus rhomboid can not use the system to screen interacted protein.Screen interacted MIC proteins with the rhomboid First E. tenella MIC1 and MIC4 gene segment were generated. Then bait plasmid pGBKT7-ROM, capture plasmid pGADT7-MIC1 and pGADT7-MIC4 were constructed. Bait and capture plasmid were cotransformed into yeast cells AH109, respectively. At last, protein interaction could be identified by X-Gal verification. The results showed that the recombinant bait plasmid and capture plasmids were constructed successfully. The target gene could be expressed in yeast cells. There was no self-activation and toxicity for yeast. Interaction of rhomboid protein and MIC4 protein was identified by Gal4 yeast two-hybrid system. Western blot showed that MIC4 protein became more smaller. We presumed that it was due to the cutting action of rhomboid proteins.Interacted verification of rhomboid and MIC4 protein First, recombinant prokaryotic expression plasmid pET28a-MIC4, recombinant eukaryotic expression plasmid pcDNA-Myc-ROM and pcDNA-HA-MIC4 were constructed. Then expression, purification and renaturation of E. tenella MIC4 were performed. Interaction of rhomboid and MIC4 was verified by western blot. The recombinant eukaryotic expression plasmids were cotransfected HeLa cells and then interaction of rhomboid and MIC4 protein was verified by immunoprecipitation. The results showed that prokaryotic and eukaryotic expression plasmids were successfully constructed. The prokaryotic expressed rhomboid protein mixed with MIC4. Immunoprecipitation showed that there was interaction between two proteins. The eukaryotic recombinant plasmids were transfected HeLa cells. Indirect immunofluorescence showed rhomboid and MIC4 protein could be successfully expressed in HeLa cells. Immunoprecipitation and western blot proved that rhomboid interacted with MIC4 protein and enzyme activity of Rhomboid protein could cut their substrate MIC4 protein. The test result provides a theoretical basi for further study of the molecular mechanisms of coccidia invasion. Protective immunity of interacted protein in yeast against E.tenella challenge Yeast cells AH109 that contained rhomboid, MIC4 and interacted protein were broken. Chickens were immunized with recombinant proteins via subcutaneous route. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, response immunity of cellular and antibody. All the recombinant proteins immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+and CD8+cells and antibody titre compared to the control(P<0.05), especially interacted protein group (P<0.01). ACI of that group could reached 168.24. The yeast group also produced a good immune protection for coccidiosis. This resule was a theoretical basis of coccidiosis vaccine research.Inhibited effect of sporozoites cell invasion was detected by DCI. E. tenella sporozoites was cultured in vitro in primary chicken kidney cells. DCI was divided into two groups. One, sporozoites were dealed with DCI, and then add into the cells. Other, DCI and sporozoites were add into the cells together. The number of the first-generation schizonts, second-generation schizonts and progeny oocysts were counted. Inhibited effect of different DCI concentrations was identified. Results showed that chicken kidney cells invasion was inhibited by 20μM DCI. At the same time, the number of progeny coccidia oocysts was controled. DCI existed potential role of treatment coccidiosis and it will be become one candidate component for the anticoccidial drugs study.更多还原