本文選題：血管內(nèi)皮生長因子121 + 骨形態(tài)蛋白2��； 參考：《遼寧醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的 構(gòu)建能夠同時在真核細(xì)胞中高表達(dá)人血管內(nèi)皮生長因子121(Vascular Endothelial Growth Factor,VEGF121)目的蛋白和人骨形態(tài)蛋白2(Bone Morphogenetic Protein , BMP2 )的新型重組腺病毒真核表達(dá)載體pAd-CMV-VEGF121-IRES-BMP2 ,并在HEK293A細(xì)胞中包裝成重組腺病毒Ad-CMV-VEGF121-IRES-BMP2。同時構(gòu)建其對照載體Ad-CMV-VEGF121-IRES-hrGFP- 1。為后續(xù)轉(zhuǎn)染兔骨髓基質(zhì)干細(xì)胞及動物體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。 方法 對目的基因供體質(zhì)粒pShuttle-CMV-BMP2攜帶的人BMP2基因進(jìn)行測序和對其序列內(nèi)部限制性內(nèi)切酶識別位點(diǎn)進(jìn)行分析,利用PCR(polymerase chain reaction,PCR)技術(shù)擴(kuò)增BMP2基因片段以及去掉其終止密碼子,之后在基因前后添加新的酶切位點(diǎn)Kpn I和Xba I,酶切、測序檢測片段序列情況。將pShuttle-CMV-VEGF121- IRES-hrGFP-1進(jìn)行測序后,使用Kpn I/Xba I雙酶切,切掉hrGFP-1片段,電泳切膠回收pShuttle-CMV-VEGF121-IRES.再將序列正確BMP2基因定向連入腺病毒穿梭載體pShuttle-CMV-VEGF121-IRES中。經(jīng)測序鑒定篩選陽性克隆,PmeⅠ酶切線性化且去磷酸化后轉(zhuǎn)化BJ5183-AD-1電感受態(tài)細(xì)胞,利用細(xì)菌內(nèi)同源重組機(jī)制將VEGF 121和BMP2基因連同其順勢表達(dá)元件重組入pAdEasy-1腺病毒系統(tǒng),通過基因測序、PCR及PacⅠ酶切鑒定獲得重組腺病毒載體pAd-VEGF121-IRES-BMP2。去內(nèi)毒素超純提取該重組腺病毒質(zhì)粒,經(jīng)PacⅠ酶切電泳后凝膠回收大片段,提純并通過脂質(zhì)體Lipofectamine 2000介導(dǎo)轉(zhuǎn)染HEK293A細(xì)胞,RT-PCR和Western blot方法檢測轉(zhuǎn)染細(xì)胞中BMP2基因和VEGF121mRNA和蛋白表達(dá)情況,完成Ad-CMV-VEGF121-IRES-BMP2重組腺病毒真核表達(dá)載體的構(gòu)建。同時重組對照Ad-CMV-VEGF121-IRES-hrGFP-1。 結(jié)果 1.經(jīng)基因測序及酶切鑒定表明,重組腺病毒真核表達(dá)載體pShuttle-CMV-VEGF121-IRES-BMP2構(gòu)建成功。 2.經(jīng)基因測序、PCR檢測及酶切鑒定表明,重組腺病毒真核表達(dá)載體pAd-VEGF121-IRES-BMP2構(gòu)建成功,同時成功構(gòu)建其對照病毒載體pAd-VEGF121-IRES-hrGFP-1。 3.轉(zhuǎn)染HEK293細(xì)胞后經(jīng)PCR檢測鑒定表明:轉(zhuǎn)染Ad-CMV-VEGF121-IRES-BMP2組(A組)與轉(zhuǎn)染Ad-CMV-VEGF121-IRES-hrGFP-1對照組(B組)細(xì)胞內(nèi)VEGF121mRNA表達(dá)量存在顯著性差異(P0.01),A組高于B組。 4.轉(zhuǎn)染HEK293細(xì)胞后Western blot結(jié)果表明:A組細(xì)胞的VEGF121蛋白表達(dá)量明顯高于B組,存在顯著性差異(P0.01)。 結(jié)論 成功構(gòu)建能夠新型重組腺病毒真核表達(dá)載體Ad-CMV-VEGF121-IRES-BMP2,為后續(xù)轉(zhuǎn)染兔骨髓基質(zhì)干細(xì)胞及動物體內(nèi)實(shí)驗(yàn)奠定基礎(chǔ)。
[Abstract]:PurposeA novel recombinant adenovirus eukaryotic expression vector pAd-CMV-VEGF121-IRES-BMP2 was constructed, which could express both the target protein of 121(Vascular Endothelial Growth factor-VEGF121 and human bone morphogenetic protein 2(Bone Morphogenetic Protein (BMP2) in eukaryotic cells. The recombinant adenovirus Ad-CMV-VEGF121-IRES-BMP2 was packaged in HEK293A cells.Meanwhile, the control vector Ad-CMV-VEGF121-IRES-hrGFP-1 was constructed.To lay a foundation for the subsequent transfection of rabbit bone marrow stromal cells and in vivo experiments.MethodThe human BMP2 gene carried by the target gene donor plasmid pShuttle-CMV-BMP2 was sequenced and the restriction endonuclease recognition sites within its sequence were analyzed. The BMP2 gene fragment was amplified by PCR(polymerase chain reaction- PCR technique and its terminating codon was removed.After that, new restriction sites Kpn I and Xba I were added before and after the gene, and the sequence of the fragments was detected by enzyme digestion and sequencing.After sequencing pShuttle-CMV-VEGF121- IRES-hrGFP-1, Kpn I/Xba I was used to cut out the hrGFP-1 fragment, and pShuttle-CMV-VEGF121-IRESwas recovered by gel electrophoresis.Then the correct BMP2 gene was inserted into the adenovirus shuttle vector pShuttle-CMV-VEGF121-IRES.The positive clone PME 鈪，

本文編號：1772499