PNP基因克隆及其表达载体的构建与鉴定

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1、摘要目的：研究PNP基因的克隆、真核表达载体的构建与鉴定，为肿瘤的基因治疗与基因工程的应用提供实验和理论依据。方法：①大肠埃希氏杆菌K12PNP基因的PCR扩增；②重组质粒pMSCVoPNP的构建；③用氯化钙法制各感受态大肠杆菌(XLl．Blue)；④转化感受态菌：用携带目的基因的重组质粒pMSCV-PNP转化感受态XLl．Blue大肠杆菌，同时设立阳性对照组和阴性对照组。转移适量转化菌于平皿培养，筛选氨苄青霉素抗性转化菌落；⑤提取质粒和酶切重组质粒：提取重组质粒pMSCV-PNP，限制性内切酶EcoRI和BglII双酶切重组质粒，电泳鉴定；⑥PCR鉴定

2、、测序鉴定重组质粒pMSCV-PNP⑦提取质粒和酶切质粒：限制性内切酶BspHl、BglII、Aft11分别酶切pVSV-G，pGAG．POL，pMSCV，电泳鉴定质粒。结果：①PNP基因PCR扩增出特异条带；②重组质粒pMSCV-PNP电泳结果与预期一致；③重组质粒pMSCV-PNP转化结果：实验组平皿上可见近百个菌落，阳性对照组有数百个菌落生长，而阴性对照组则未见菌落生长；④阳性克隆质粒pMSCV-PNPPCR鉴定、酶切鉴定，电泳条带与预期一致；测序结果正常；⑤克隆质粒pVSV-G，pGAG．POL，pMSCV酶切鉴定与预期一致。结论：①从大肠埃希氏

3、杆菌K12中克隆到PNP基因；②构建了表达载体pMSCV-PNP；③抽提了三种载体质粒pMSCV、pVSV-G、pGAG—POL。关键词：PNP克隆；重组质粒；鉴定IIAbs仃actABSTRACTobjeetive：ToconstructandidentifytherecombinantvectorpMSCV-PNPcarryingE．coliK12PNPwhichCanexpressineukaryotecellsandwhichwillprovidethebasisforgenetherapy．Methods：①PNPwereamplifiedfr

4、omE．coliK12bacteriabypolymerasechainreaction(PCR)．②ToconstructtherecombinantvectorpMSCV-PNPbymeansofT4一DNAligase．③CompetentEcoli(strainXLl一Blue)waspreparedusingcalciumchloroid．④Transformationofthecompetentbacteria：WetransformedthecompetentbacteriawiththeplasmidspVSV-G,pGAG-POL，pM

5、SCVandpMSCV-PNPcontainingPNPgene．TheappropriatevolumeofthetransformedbacteriawastransferedandspreadedontoagarLBmediumtoselecttheampicillin．resistantcolonies．⑤Extractionandrestrictionendonucleaseanalysisoftheplasmids：TheplasmidspVSV-GpGAG—POL，pMSCV,pMSCV-PNPwereextractedfromthetra

6、nsformedbacteriawithplasmidpurificationkit，andtheywerecutwithrestrictionendonucleasesBspHI、BglII、AftII，EcoRIandBgl兀，respectively．Results：①Ampicillinresistanttranformedbacteriacoloniesgrowth：AftertransformationwithpMSCV，pMSCV-PNP,pGAG—POLandpVSV-G,about100colonieswereobservedonthe

7、platecontainingampicillin，andthecolonieswerenotfoundinthenegativecontrolgroups．②Thenormalsizeofthefragmentsfromtheplasmidscutbyendonucleases：TheresultsofrestrictionendonucleaseanalysisandagarelectrophoresisindicatedthatsizeofplasmidspMSCV,pMSCV-PNP,pGAG-POLandpVSV-GWasnormal．Conc

8、lusion：E．coliK12PNPCanbesuccessfullyclon