【作者】 吴启进；

【导师】 石火英； 金亚美；

【作者基本信息】 扬州大学 ， 基础兽医学， 2010， 硕士

【摘要】 血吸虫病是一种在世界范围内广泛分布,由血吸虫感染引起的危害严重的人畜共患寄生虫病。血吸虫为吸虫中独特的雌雄异体形式,雌雄虫的合抱是血吸虫的生长发育的关键,抱雌沟蛋白是目前发现的唯一一种在雌雄虫合抱过程中进行传递的蛋白质。所以对抱雌沟蛋白及其相互作用蛋白的研究,对了解血吸虫生长发育的机理具有重要的意义一、抱雌沟蛋白相互作用因子的筛选利用本实验室构建的诱饵蛋白-血吸虫抱雌沟蛋白重组质粒pGBKT7-BD-SjGCP与成功构建的18日龄日本血吸虫酵母双杂交cDNA预转化文库进行抱雌沟蛋白相互作用因子的筛选。对筛选出的383个阳性文库质粒的外源插入片段进行序列测定得到131个外源插入序列,对其进行生物信息学分析,除去重复序列,共得到45个不同的有效基因序列。将得到的基因序列重新构建到pGADT7-AD载体上,与pGBK-BD-SjGCP进行回复杂交验证其相互作用,去除假阳性后得到了16个与血吸虫抱雌沟蛋白相互作用的蛋白的基因序列。本试验在筛选过程中采取了反复传代,并对阳性克隆进行回复杂交验证,保证了假阳性的去除,得到的16个蛋白经分析表明其有充分的与抱雌沟蛋白作用的可能性,但这些相互作用尚需进一步采取其他方式验证。二、日本血吸虫Actin及Twinfilin蛋白的初步研究为研究酵母双杂交所筛选出与SjGCP相互作用的SjACT和SjTWF蛋白,本文首先构建了这两种蛋白的重组表达载体,并在大肠杆菌中获得大量的原核表达产物。Westren-blot方法证实了表达出的蛋白具有良好的免疫原性,并且发现筛选出的SjActin蛋白为肌动蛋白家族中的Smooth Muscle Actin(SMA,Actin-α2)蛋白。利用抗SMA蛋白的单抗及SjGCP的多抗进行的激光共聚焦荧光共定位实验表明这两种蛋白在空间上都定位在血雌虫表膜上,从而证实了酵母双杂交筛选的可靠性。由Real-time方法分析SjActin和SjTWF在血吸虫发育不同时期的表达情况时发现SjActin和SjTWF的转录分别在血吸虫感染宿主后的23和21天较高。对SjTWF蛋白的免疫反应类型分析发现,免疫鼠脾细胞产生的TNF-α和IFN-γ量在SjTWF刺激后明显升高,差异极显著(P<0.001),而脾细胞产生的IL-10量在SjTWF刺激前后都比较高,之间没有显著差异(P>0.05),产生IL-4的量在SjTWF刺激后升高(P<0.05)。这说明SjTWF即刺激机体产生ThⅠ类型的免疫反应,也刺激机体产生ThⅡ类型的免疫反应。三、RNA干扰日本血吸虫Actin, Twinfilin蛋白的研究为更进一步确定SjACT和SjTWF在血吸虫生长发育过程中所起的作用,本文分别设计三对小RNA分子干扰体外培养血吸虫的SjACT和SjTWF蛋白表达。对干扰前后SjACT和SjTWF基因转录水平和蛋白表达水平的分析发现干扰获得成功,且发现干扰效果最佳的小分子干扰该蛋白的表达引起较高的血吸虫死亡率。对小RNA分子干扰对血吸虫形态影响的观察发现SjACT和SjTWF蛋白表达的下降都会影响血吸虫的运动,结果表明sjACT和sjTWF具有影响血吸虫运动和生长的作用。更多还原

【Abstract】 Schistosomiasis is a serious parasitic disease infected by schistosome, which infects people and livestock and widely distributed in the world. Schistosome is a unique dioecious fluke, SGCP is the only protein which was found expressed especially at the gynaecophoral canal of male worms and transfer from male onto female, and SGCP was thought have some function on the development and mature of female worms. So the studies on SGCP and proteins interacted with SGCP would show some light on the mechanism of growth and development of schistosome.一.Screening interactional factor of SjGCP proteinRecombinant plasmid pGBKT7-BD-SjGCP and 18d of Schistosoma japonicum cDNA library have been constructed ready for yeast Two-hybrid system. To screen of protein interact with SjGCP. , 131 inserted fragments was acquired from 383 positive recombinant plasmid, sequencing analysis of these fragments show that 45 different genes sequence we have got. Then these genes have been cloned into the plasmid of pGBKT7-BD-SjGCP to verify interaction of SjGCP protein by repeating Two-Hybrid, also Negative Control is done in order to remove false positive. Finally 16 genes sequence of the protein which interact with SjGCP were finally got. Because of using repeat subcultured yeast cell and repeat Two- Hybrid to remove false positive ,the results of 16 protein have be screened is reliable in this study .二.SjACT and SjTWF protein preliminary researchIn order to study of SjACT and SjTWF protein, screened by yeast Two-Hybrid. First, Recombinant plasmids of pET28a-SjACT and pET28a-SjTWF were constructed and SjACT and SjTWF proteins were expressed in E. coli. Good immunogencity was be verified using antiserum by western-bolt method, this results also confirmed that SjACT is a Smooth Muscle Actin (SMA,Actin-α2) of actin protein family. Then, sjACT and sjGCP protein was observed expression upper Schistosoma japonicum epidermis by using Laser Scanning Confocal Microscope, so this study shows reliability of interaction of SjGCP and SjACT protein. Additionally, Transcript level of SjACT and sjTWF is highest on 23d, 21d was detected out individually by using real-time PCR method. Finally we detected IL-10、TNF-α、IFN-γand IL-4 production following splenocyte activation with SjTWF protein,TNF-αand IFN-γproduction shows Significant differences from stimulated in relation to non-stimulated cells( P<0.001), but IL-10 production shows no significant differences (P>0.5), and IL-4 shows differences(P<0.5).This finding suggests a tendency of Th1 and Th2 type of immune response.三. RNA interference SjACT and SjTWF proteinTo determine the role of SjACT and SjTWF in Schistosoma japonicum growth and development, three siRNA was design to interference of SjACT and SjTWF protein expression on japonicum cultivated in vitro. analysising from transcriptional level and production of protein express, this experiment was succeed, and founding mortality of japonicum have positive correlation to effect of interference by siRNA. Observation form of Morphology, movement affected when interference of SjACT and SjTWF protein expression of japonicum. Conclusion that SjACT and SjTWF protein have some relationship to Schistosoma japonicum growth and development.更多还原