人snca基因真核表達(dá)載體的構(gòu)建及其在pc12細(xì)胞中的表達(dá)

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1、人SNCA基因真核表達(dá)載體的構(gòu)建及其在PC12細(xì)胞中的表達(dá)作者：錢進(jìn)軍,程言博,劉春風(fēng),楊亞萍,劉康永【摘要】　　目的:構(gòu)建含人野生型及致病突變A30P、A53TSNCA基因的重組真核表達(dá)載體pEGFPC3SNCA,并通過穩(wěn)定轉(zhuǎn)染獲得過表達(dá)人野生型及致病突變A30P、A53Tαsynuclein(SNCA)的單克隆PC12細(xì)胞株。方法:通過RTPCR方法擴(kuò)增SNCA基因,TA克隆后測序。在此基礎(chǔ)上利用單核苷酸差異引物定點(diǎn)誘變法相繼構(gòu)建含SNCA基因編碼區(qū)EcoRI、BamHI兩酶切位點(diǎn)的同義突變及其致病突變G88C（Ala30

2、Pro）、G209A（Ala53Thr）的重組真核表達(dá)載體pEGFPC3SNCA,并以PCR、雙酶切、測序鑒定。以重組質(zhì)粒pEGFPC3SNCA通過脂質(zhì)體轉(zhuǎn)染方法轉(zhuǎn)染PC12細(xì)胞;以G418進(jìn)行篩選;以有限稀釋法進(jìn)行亞克隆獲得穩(wěn)定過表達(dá)人野生型及致病突變A30P、A53Tαsynuclein的單克隆PC12細(xì)胞株,并以穩(wěn)定轉(zhuǎn)染pEGFPC3的PC12細(xì)胞作為對照組細(xì)胞,通過RTPCR、Westernblot及熒光顯微鏡鑒定各單克隆PC12細(xì)胞株。結(jié)果:PCR、酶切及測序證明重組真核表達(dá)載體pEGFPC3SNCA構(gòu)建成功

3、。RTPCR、Westernblot及熒光顯微鏡確認(rèn)目的基因序列在PC12細(xì)胞穩(wěn)定過表達(dá)。結(jié)論:成功地構(gòu)建SNCA基因WT及A30P與A53T突變型的重組真核表達(dá)載體pEGFPC3SNCA;成功獲得過表達(dá)人野生型及致病突變A30P、A53Tαsynuclein的單克隆PC12細(xì)胞株。14【關(guān)鍵詞】αsynuclein（SNCA）致病突變真核表達(dá)載體pEGFPC3穩(wěn)定轉(zhuǎn)染PC12細(xì)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