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ID:33202174
大小:9.75 MB
页数:84页
时间:2019-02-22
《广西猪瘟病毒分子流行病学调查及单克隆抗体的制备》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、广西大学硕士学位论文广西猪瘟病毒分子流行病学调查及单克隆抗体的制备姓名:毛燕申请学位级别:硕士专业:预防兽医学指导教师:吴健敏20120628广西猪瘟病毒分子流行病学调查及单克隆抗体的制备摘要本研究采集CSFV疑似样品并利用巢氏RT-PCR方法进行检测,从检测CSFV核酸阳性样品中挑选有代表性的进行E2基因克隆。将获得的序列与在genebank登录的其它广西CSFVE2基因序列进行综合分析,全面了解广西CSFV流行及遗传变异情况。从结果中发现广西流行毒株与HCLV疫苗株氨基酸与核苷酸同源性均随着时间推移发生变化
2、,核苷酸、氨基酸最高均下降了3.7%。通过构建进化树发现,广西流行毒株属于2个基因群,4个基因亚型,即1.1和2.1、2.2、2.3亚群,基因2.1亚型毒株已成为流行的优势毒株,未见基因1.2亚型及基因3群毒株。再通过统计数据发现保育猪发病仍然占第一位,但哺乳仔猪发病率呈上升的趋势,由过去的18.42%提高到32.39%。以SHJMEN株为代表的1.1亚型毒株仍然存在广西猪群中,主要引起母猪繁殖障碍和隐性感染,新生仔猪发病为主,基因2.1型毒株主要引起保育猪发病。本研究通过CSFV分子流行病学调查,掌握广西近年
3、来猪瘟病毒的流行情况,为制定广西猪瘟的防控措施提供依据。此外,将本实验室保存的猪瘟病毒经纯化后免疫BALB/c小鼠,通过单克隆抗体技术制备了两株长期稳定分泌抗猪瘟病毒的单抗,分别命名为CSFV7和CSFVl4。对单抗进行鉴定后发现,CSFV7和CSFVl4杂交瘤培养上清效价为1:256,腹水上清效价达l:106;抗体亚类为IgG2b;相对亲和指数分别为1.5mol/L、lmoi/L,表明其有较好的亲和力;单抗TWestern—blot与IFA反应证实两株单抗不仅特异性与CSFV反应,而且是针对猪瘟病毒E2重组蛋
4、白的单克隆抗体。制各灵敏度高、特异性强的抗猪瘟病毒单克隆抗体,为进一步建立快速检测猪瘟病毒抗原方法打下基础。关键词广西猪瘟病毒分子流行病学单克隆抗体MOLECULAREPIDEⅣⅡ0LOGICALSIⅡ5、heCSFVsuspectedsamplesandnestedRT-PCRmethodfordetection,detectionofCSFVnucleicacidsamples。WeselectandCloneCSFVrepresentativestrainofE2.ByComprehensiveanalysisCombiningtheobtainedsequenceandpandemicstrainsequenceofCSFVinGuangxidownloadingfromGnenBank,itletUSt6、ocomprehensiveunderstandingofGuangxitheCSFVpopularandgeneticvariationsituation.Fromtheresults,WefoundAminoacidandnucleotidehomologyoftheGuangxipandemicstrain、HCLVvaccinestrainstheandSHIMENstrainstochangeovertime.thenucleotideandaminoacidsthehighestdecreasedb7、y3.7%.ByconstructingThephylogenetictreeofGuangxiCSFVpandemicstrainandmeybelongtothetwogenes,4genotype,itcontain1.1and2.1,2.2,2.3subsetsgene.2.1subtypehasbecomeapopularstrainoftheadvantages,nogene1.2subtypesand3-typestrain.ByStatisticaldata,conservationofpigd8、iseasestillaccountsforthefirst,butthepigletsincidencerateshowedanupwardtrendandincreasedto32.39percentfrom18.42percentinthepast.1.1111subtypeSHIMENstrainsstillexistintheGuangxipigs,themaincauseo
5、heCSFVsuspectedsamplesandnestedRT-PCRmethodfordetection,detectionofCSFVnucleicacidsamples。WeselectandCloneCSFVrepresentativestrainofE2.ByComprehensiveanalysisCombiningtheobtainedsequenceandpandemicstrainsequenceofCSFVinGuangxidownloadingfromGnenBank,itletUSt
6、ocomprehensiveunderstandingofGuangxitheCSFVpopularandgeneticvariationsituation.Fromtheresults,WefoundAminoacidandnucleotidehomologyoftheGuangxipandemicstrain、HCLVvaccinestrainstheandSHIMENstrainstochangeovertime.thenucleotideandaminoacidsthehighestdecreasedb
7、y3.7%.ByconstructingThephylogenetictreeofGuangxiCSFVpandemicstrainandmeybelongtothetwogenes,4genotype,itcontain1.1and2.1,2.2,2.3subsetsgene.2.1subtypehasbecomeapopularstrainoftheadvantages,nogene1.2subtypesand3-typestrain.ByStatisticaldata,conservationofpigd
8、iseasestillaccountsforthefirst,butthepigletsincidencerateshowedanupwardtrendandincreasedto32.39percentfrom18.42percentinthepast.1.1111subtypeSHIMENstrainsstillexistintheGuangxipigs,themaincauseo
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