摘要
目的构建融合表达Fas死亡信号激发域(Fasactivationdomain,FasAD)的重组载体FasAD-pTYB2。方法应用半巢式逆转录-多聚酶链反应(RT-PCR)技术克隆Fas死亡信号激发域cDNA片段FasAD,插入通用载体pGEM-T中鉴定,然后装入Intein表达型原核载体pTYB2中,经IPTG(isopropylthio-β-D-galactoside)诱导表达融合蛋白。结果DNA序列测定显示克隆的cDNA片段序列正确。初步获得融合蛋白的表达,经Western-blotting鉴定,融合蛋白具有良好的Fas抗原性。结论上述结果提示可利用该表达型重组质粒制备人Fas死亡信号激发域多肽/intein的融合蛋白。
Objective To constructtherecombinantplasmidFasAD-pTYB2forthefusionexpressionof Fasactivationdomain(FasAD).Methods FasADcDNAwasclonedby semi-nestedreversetranscriptase-polymerasechainreaction(RT-PCR),and theninsertedintotheuniversalvectorof pGEM-Tfortheidentificationof itsDNAsequence.ThetargetDNAfragmentwas insertedintotheprokaryotevectorpTYB2thatexpressedinteinto constructtheplasmidrecombinantFasAD-pTYB2which wasinducedby isopropylthio-β-D-galactosidefor the expressionof the fusionprotein.Results DNA sequenceanalysis confirmedsequenceof FasADcDNApreviouslycloned,andexpressionof thefusionproteinby therecombinantplasmidwas achieved,the productrecognizableby rabbitanti-humanFas polyclonalantibodyas demonstratedby Westernblotting analysis.Conclusion TherecombinantplasmidFasAD-pTYB2constructedin thisstudyis capableof expressingthefusion proteinof FasADandintein,whichmaybe significantforfurtherstudyof theepitopeandfunctionof Fasantigen.
出处
《第一军医大学学报》
CSCD
北大核心
2002年第3期227-230,共4页
Journal of First Military Medical University
基金
广东省自然科学基金(1999-2001)
