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1、摘要目的：研究PNP基因的克隆、真核表達載體的構建與鑒定，為腫瘤的基因治療與基因工程的應用提供實驗和理論依據。方法：①大腸埃希氏桿菌K12PNP基因的PCR擴增；②重組質粒pMSCVoPNP的構建；③用氯化鈣法制各感受態(tài)大腸桿菌(XLl．Blue)；④轉化感受態(tài)菌：用攜帶目的基因的重組質粒pMSCV-PNP轉化感受態(tài)XLl．Blue大腸桿菌，同時設立陽性對照組和陰性對照組。轉移適量轉化菌于平皿培養(yǎng)，篩選氨芐青霉素抗性轉化菌落；⑤提取質粒和酶切重組質粒：提取重組質粒pMSCV-PNP，限制性內切酶EcoRI和BglII雙酶切重組質粒，電泳鑒定；⑥PCR鑒定
2、、測序鑒定重組質粒pMSCV-PNP⑦提取質粒和酶切質粒：限制性內切酶BspHl、BglII、Aft11分別酶切pVSV-G，pGAG．POL，pMSCV，電泳鑒定質粒。結果：①PNP基因PCR擴增出特異條帶；②重組質粒pMSCV-PNP電泳結果與預期一致；③重組質粒pMSCV-PNP轉化結果：實驗組平皿上可見近百個菌落，陽性對照組有數百個菌落生長，而陰性對照組則未見菌落生長；④陽性克隆質粒pMSCV-PNPPCR鑒定、酶切鑒定，電泳條帶與預期一致；測序結果正常；⑤克隆質粒pVSV-G，pGAG．POL，pMSCV酶切鑒定與預期一致。結論：①從大腸埃希氏
3、桿菌K12中克隆到PNP基因；②構建了表達載體pMSCV-PNP；③抽提了三種載體質粒pMSCV、pVSV-G、pGAG—POL。關鍵詞：PNP克??；重組質粒；鑒定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
