搜索
学号：071040132本科毕业论文患病鲫肾组织中病毒解旋酶基因的检测院（系）：生物科学与技术学院专业：生物工程姓名：李涛班级：0710401指导教师：周毅峰副教授中国·恩施二〇一四年五月StudentID：071040132BACHELOR'STHESISOFHUBEIUNIVERSITYFORNATIONALTIESDetectiontheherpesviralhelicasegenefromthediseasedgibelcarpCarassiuscarassiusName：TaoLiGrade：0710401Profession：BiologicalengineeringTutor：Coprof.Yi-FengZhouEnshi•ChinaMay,2014学术声明1、始终以“求真、务实、创新”的科学研究态度从事各项研究工作。2、在本论文中，除了已经标注的所有引用文献以为，其他所有均为自己试验真实结果。3、在本论文中除了已经标注了的引用文献外，没有包含任何人或者机构的科研成果。4、对本论文作出帮助和贡献的同志，在文章中做了声明并感谢。5、本声明的法律后果由本人承担。作者签名：年月日导师审核声明本人郑重声明：该学生所写学士学位毕业论文，是在本人的指导下完成。其中文字、图、表及其它相关内容均经本人审核，同意作为该生的学位论文提交。导师签名：年月日患病鲫肾组织中病毒解旋酶基因的检测李涛(湖北民族学院生物科学与技术学院，恩施，445000)摘要：为查明部分养殖场鲫患出血病的病原，结合体表症状、病变组织及鱼疱疹病毒基因序列，进行了形态解剖、PCR及RT-PCR检测以及病毒回接感染，初步分析了引起鲫出血病的病原。结果表明：三十尾患病鲫体表，尤其是腹部出现点状出血点，解剖后观察到肝脏、肾脏、脾都有充血型肿胀，肠肿胀变粗。PCR扩增患病鲫基因组DNA和RT-PCR扩增患病鲫肾转录组均出现一条约360bp特异性片段，和预测362bp片段大小基本相符，证实了该患病鲫肾中含疱疹病毒解旋酶基因，回接感染三天左右鱼腹部开始出现点状出血点，四天左右鱼开始死亡。因此，推测疱疹病毒与鲫出血病有关原因之一，其它因素是否与鲫出血病有关有待进一步实验证实。关键词：鲫；鱼疱疹病毒；PCR；RT-PCR；出血病DetectiontheherpesviralhelicasegeneformthediseasedgibelcarpCarassiuscarassiusTaoLi(Schoolofbiologicscienceandtechnology,Hubeiuniversityfornationalities,Enshi,445000)Abstract：InordertoindentifythecausativepathogenofthediseasewithseverehemorrhageshasemergedinsomefarmsofcentralChina.Thegrosspathologicalsignswereobservedandobservationoftheexternalsurfaceoftensofdiseasedgibelcarpshowedseveredottedhemorrhagesonthebodysurface,especiallyontheabdomen.Anatomyinspectionshowedthatliver,kindey,spleenandintestineallpresentedsymptomsofswelling.ThetargetgenewasamplifiedwiththedesignedprimerbythePCRmethod.ThePCRproductwasanalyzedbyagarosegelelectrophoresisandaspecializedfragmentwasobservedwith360bp,whichmatchedthesizeofthetargetgeneabout362bp,indicatingthatthegeneofhelicaseofherpesvirusexistedinthekidneyofdiseasedfish.Tiebackinfectedfishbellybegantoappearaboutthreedaysspottingpoints,aboutfourdaysthefishbegantodie.ThepresentstudyshowedthattheDNAandRNAofhelicsaseoffishherpesvirusfromdiseasedfishhavebeendetected,andthisresultindicatedthatfishherpesviruswasrelatedtothediseaseofhemorrhagesfordiseasedgibelcarp.However,furtherstudiesareneededtodemonstratethisresult.Keywords：Gibelcarp;Hemorrhagicdisease;Fishherpesvirus;PCR;RT-PCR目录1引言.........................................................................................................21.1研究背景.............................................................................................................21.2鱼病毒病检测方法...........................................................................................21.2.1细胞培养检测...........................................................................................21.2.2分子生物学检测.......................................................................................21.3疱疹病毒.............................................................................................................31.3.1疱疹病毒的形态和种类..........................................................................31.3.2鱼和水产动物疱疹病毒...........................................................................31.3.3鱼感染疱疹病毒.......................................................................................41.4鲫出血病...........................................................................................................41.4.1病毒性出血病和细菌性出血病的区别...................................................41.4.2出血病的防治...........................................................................................41.5本研究的目的与意义........................................................................................52材料与方法...............................................................................................62.1材料.....................................................................................................................62.2试剂与设备.........................................................................................................62.2.1试剂...........................................................................................................62.2.2主要设备...................................................................................................62.3方法.....................................................................................................................62.3.1DNA的提取...........................................................................................62.3.2PCR扩增.................................................................................................72.3.3PCR引物的设计.....................................................................................72.3.4RNA提取................................................................................................72.3.5RNA逆转录............................................................................................82.3.6RT-PCR扩增...........................................................................................92.3.7琼脂糖凝胶电泳检测...............................................................................92.3.8病毒的回接感染................................................................................