人snca基因真核表达载体的构建及其在pc12细胞中的表达

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1、人SNCA基因真核表达载体的构建及其在PC12细胞中的表达作者：钱进军,程言博,刘春风,杨亚萍,刘康永【摘要】　　目的:构建含人野生型及致病突变A30P、A53TSNCA基因的重组真核表达载体pEGFPC3SNCA,并通过稳定转染获得过表达人野生型及致病突变A30P、A53Tαsynuclein(SNCA)的单克隆PC12细胞株。方法:通过RTPCR方法扩增SNCA基因,TA克隆后测序。在此基础上利用单核苷酸差异引物定点诱变法相继构建含SNCA基因编码区EcoRI、BamHI两酶切位点的同义突变及其致病突变G88C（Ala30

2、Pro）、G209A（Ala53Thr）的重组真核表达载体pEGFPC3SNCA,并以PCR、双酶切、测序鉴定。以重组质粒pEGFPC3SNCA通过脂质体转染方法转染PC12细胞;以G418进行筛选;以有限稀释法进行亚克隆获得稳定过表达人野生型及致病突变A30P、A53Tαsynuclein的单克隆PC12细胞株,并以稳定转染pEGFPC3的PC12细胞作为对照组细胞,通过RTPCR、Westernblot及荧光显微镜鉴定各单克隆PC12细胞株。结果:PCR、酶切及测序证明重组真核表达载体pEGFPC3SNCA构建成功

3、。RTPCR、Westernblot及荧光显微镜确认目的基因序列在PC12细胞稳定过表达。结论:成功地构建SNCA基因WT及A30P与A53T突变型的重组真核表达载体pEGFPC3SNCA;成功获得过表达人野生型及致病突变A30P、A53Tαsynuclein的单克隆PC12细胞株。14【关键词】αsynuclein（SNCA）致病突变真核表达载体pEGFPC3稳定转染PC12细胞　　[Abstract]AIM:ToconstructrecombinanteukaryoticexpressionvectorspEGFPC3S

4、NCAcontaininghumanwildtype(WT)andpathogenicmutationsA30P,A53Tαsynuclein(SNCA),andtoobtainmonoclonalPC12celllinesoverexpressinghumanwildtypeandpathogenicmutationsA30P,A53Tαsynucleinbystabletransfection.METHODS:HumanwildtypeSNCAgenewasclonedbyusingRTPCR.ByTAextension

5、cloning,thegenewasligatedwithTvectorandsequenced.Basedonit,therecombinanteukaryoticexpressionvectorscontainingwildtypeSNCAsynonymousmutationoritsG88C(Ala30Pro)andG209A(Ala53Thr)pathogenicmutationwereconstructedbysitedirectedmutagensisusingprimervarianceinmononucleotide

6、.AnddifferentrecombinantplasmidspEGFPC3SNCAwereidentifiedwithPCR,restrictionenzymedigestionandDNAsequencing.PC12cellsweretransfectedwithdifferentrecombinantplasmidspEGFPC3SNCAbyliposometransfectionmethod,screenedwithG418,andsubclonedbylimiteddilutionmethodtoobtaindif

7、ferentmonoclonalPC12celllinesstably14overexpressinghumanwildtype,A30PorA53Tαsynuclein,respectively,andPC12cellsstablytransfectedwithpEGFPC3wereusedascontrolgroup.ThesemonoclonalPC12celllineswereidentifiedwithRTPCR,Westernblotandfluorescencemicroscopy.RESULTS:Accordi

8、ngtoresultofPCR,thedoubledigestionandgenesequencing,itwascomfirmedthatrecombinanteukaryoticexpressionve