havp2重组杆状病毒的构建与其表达

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1、一●黑龙江大学硕士学位论文rBac．TZF．WK—HA．VP2、rBac．TZF．WK．I．HA．VP2、rBac．TZF．SD．HA．VP2、rBac．TZF．SD—I-HA-VP2、rBae．TZF．LM。HA．VP2、rBac-TZF．LM．I．HA．VP2、rBae．TZF．WKC．HA-VP2、rBae-TZF．SDC．HA-VP2、rBae-TZF．LMC-HA-VP2，转染鼍汐昆虫细胞后，获得重组杆状病毒P1代储备，继续扩增病毒至P3代，空斑法分析病毒的滴度均在1．2×108pfu／mL左右。最后，将获得

2、的9个重组杆状病毒同时侵染中国仓鼠卵巢细胞(CHO)，利用SDS—PAGE和Western．blotting成功检测到HA-VP2蛋白的表达。研究结果表明，优化后的杆状病毒表达载体系统能够较好地表达具有抗原性的传染性法氏囊病病毒VP2抗原蛋白，为探究改进型重组杆状病毒作为基因传递的载体研制禽类基因工程疫苗奠定了坚实的基础。关键词：传染性法氏囊病病毒(mDV)；VP2；杆状病毒表达载体系统一IAbstractInfectiousbursaldiseasevirus(Infectiousbursaldiseasevirus

3、，IBDV)isacuteandhighlycontagiousdiseasepathogenswhichcancausediseaseinchickenandturkey．VP2isthemajorprotectiveantigenofhost．VP2concemes丽也thestrengthofIBDVvirulence，antigenicvariationandvirusneutralizingantibodyinductionandidentify．Themajordirectionofthepreventio

4、nandcontrolofIBDresearchisexploitingtheexpressionvectortoexpressVP2usedtopreparegeneticallyengineeredvaccine．Baculovirusexpressionvectorsystem(BaeulovirusExpressionVectorSystem,BEVS)whoseforeigngenesexpressionvectorisabaculovirus，andwhosereceptorisinsectcell，isa

5、safe，efficientvectorsystemwithlargecapacity．Itiseasytoscreenrecombinantvirus，andtherecombinantproteincouldbefoldedandmodifiedwithbiologicalactivityandSOon．Epitopetagging(epitopetaDistheattachedepitopefusetodetectproteinSOthatcanbeusedtodetecttheexpressionofprote

6、in,andalSOcanbeusedtopurifythetargetproteinbyaffinitychromatographyortolocatesubcellularproteinsbyimmunofluorescence．HA-tagisthemorewidelyusedepitopetagging．ThisissuefusedHA-TAGandIBDV-VP2，andusedBae-to-Baebaculovirusexpressionvectorsystemtoexpressthefusionprote

7、ininCHOceUlines．Comparedandanalyzedtheinfluenceofthelevelofproteinexpressionindifferentrecombinantvector，thenchosethepreferredexpressionsystem．Thisissueprovidesnewideasforpreparingthegeneticengineeringofpoultryvaccine．UsingDNAstarsoftwaretodesignapairofspecificp

8、rimersoftheVP2thatrefertopublishedIBDV—VP2sequenceonGenBank，andfusedtheHA—TAGgenetotheIBDV-VP2genesequenceofthe5’end．HA-VP2genewasamplifiedbyPa艮andclonedintopMD18·Tve