本文選題：慢病毒 + p55PIK��； 參考：《華中科技大學(xué)》2011年碩士論文

【摘要】：目的: 構(gòu)建p55PIK慢病毒表達(dá)載體Lenti-p55PIK,并觀察高表達(dá)p55PIK對(duì)腫瘤細(xì)胞增殖的影響。 方法: 用PCR的方法擴(kuò)增得到人p55PIK編碼區(qū)全長(zhǎng)片段,通過(guò)酶切、連接、轉(zhuǎn)化和質(zhì)粒提取等步驟得到重組慢病毒表達(dá)質(zhì)粒p55PIK-pCDF,并進(jìn)行酶切和基因測(cè)序鑒定。脂質(zhì)體轉(zhuǎn)染法在293FT細(xì)胞中轉(zhuǎn)染pCDF-p55PIK、pFIV與pVSVG,包裝得到可以產(chǎn)生綠色熒光的慢病毒Lenti-p55PIK。將慢病毒Lenti-p55PIK感染腫瘤細(xì)胞,用Western blot法檢測(cè)p55PIK的表達(dá),用流式細(xì)胞技術(shù)檢測(cè)增殖細(xì)胞相關(guān)抗原Ki67的表達(dá),觀察p55PIK對(duì)細(xì)胞增殖的影響。 結(jié)果: PCR產(chǎn)物進(jìn)行瓊脂糖凝膠電泳,得到1400bp左右的條帶,大小與p55PIK編碼基因序列大小一致。重組p55PIK-pCDF質(zhì)粒進(jìn)行酶切初步鑒定,證明了p55PIK編碼基因序列插入到了pCDF質(zhì)粒中。經(jīng)構(gòu)建好的質(zhì)粒經(jīng)酶切初步鑒定后送測(cè)序,結(jié)果完全符合p55PIK編碼基因序列。將pCDF-p55PIK、pFIV與pVSVG轉(zhuǎn)染293FT細(xì)胞進(jìn)行包裝,熒光顯微鏡下可以觀察細(xì)胞包裝效率,裂解細(xì)胞得到的病毒Lenti-p55PIK。將病毒感染腫瘤細(xì)胞,在熒光顯微鏡下觀察到細(xì)胞內(nèi)顯示的綠色熒光,Western blot法檢測(cè)p55PIK的表達(dá),Lenti-p55PIK組顯著高于空病毒組和空白對(duì)照組,(P0.01),流式細(xì)胞技術(shù)檢測(cè)增殖細(xì)胞相關(guān)抗原Ki67的表達(dá),結(jié)果顯示Lenti-p55PIK組顯著高于空病毒組和空白對(duì)照組,(P0.05)。 結(jié)論: 成功構(gòu)建了人p55PIK慢病毒表達(dá)載體Lenti-p55PIK,其能夠在腫瘤細(xì)胞中高表達(dá)人p55PIK蛋白,而且觀察到p55PIK促進(jìn)增殖細(xì)胞相關(guān)抗原Ki67的表達(dá)。
[Abstract]:Aim: to construct p55PIK lentivirus expression vector Lenti-p55PIK and to observe the effect of p55PIK overexpression on the proliferation of tumor cells. Methods: the full-length fragment of human p55PIK coding region was amplified by PCR. The recombinant lentivirus expression plasmid p55PIK-pCDFwas obtained by enzyme digestion, ligation, transformation and plasmid extraction, and identified by enzyme digestion and gene sequencing. PCDF-p55PIKPFIV and pVSVG were transfected into 293FT cells by liposome transfection, and the lentivirus Lenti-p55PIK, which could produce green fluorescence, was packaged. Lenti-p55PIK was infected with Lenti-p55PIK. The expression of p55PIK was detected by Western blot, and the expression of Ki67 was detected by flow cytometry. The effect of p55PIK on cell proliferation was observed. Results: agarose gel electrophoresis showed that the 1400bp band was about the same size as p55PIK coding gene. The recombinant p55PIK-pCDF plasmid was identified by restriction endonuclease digestion. It was proved that p55PIK coding gene sequence was inserted into pCDF plasmid. The constructed plasmids were identified and sequenced by restriction endonuclease digestion, and the results were in good agreement with p55PIK coding gene sequence. PCDF-p55PIKPFIV and pVSVG were transfected into 293FT cells for packaging. The packaging efficiency of the cells and the virus Lenti-p55PIK were observed under fluorescence microscope. The expression of p55PIK in the tumor cells infected with virus was detected by Western blot method under fluorescence microscope. The expression of p55PIK in the Lenti-p55PIK group was significantly higher than that in the empty virus group and the blank control group (P0.01). The expression of Ki67 was detected by flow cytometry. The results showed that Lenti-p55 PIK group was significantly higher than that of empty virus group and blank control group (P0.05). Conclusion: human p55PIK lentivirus expression vector Lenti-p55PIK was successfully constructed, which can overexpress human p55PIK protein in tumor cells, and the expression of p55PIK associated antigen Ki67 was observed.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R373

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相關(guān)期刊論文 前4條

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本文編號(hào)：2068270