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  本文選題：MxA　切入點：NIH-3T3細胞　出處：《蘇州大學》2008年碩士論文　論文類型：學位論文

【摘要】： 構建人的抗流感病毒基因-MxA(Myxovirus resistance protein A)克隆真核表達載體pEGFP-C1,再將pEGFP-C1-MxA轉染NIH-3T3細胞,通過G418抗性結合綠色熒光篩選出雙陽性細胞克隆,用本室制備的鼠抗MxA蛋白血清,對上述的細胞克隆進行Immunoblot分析和細胞免疫熒光/細胞免疫化學分析,并用RT-PCR技術對上述細胞中的MxA特異性序列進行分析鑒定,再用標準VSV病毒株攻擊進行抗病毒效應試驗。結果顯示:pEGFP-C1-MxA轉染的NIH-3T3細胞經G418抗性和綠色熒光蛋白篩選并經二輪亞克隆篩選了3個雙陽性細胞克隆(1.4.5,4.3.6,3.2.16)和EGFP基因轉染克隆;以MxA基因與鼠Mxl基因非同源區(qū)的序列設計的特異的引物進行RT-PCR試驗三株均顯陽性,Immunoblot分析三株細胞表現強陽性,免疫細胞化學分析顯示上述三株細胞有大于98%的細胞表現為強陽性。VSV抗性試驗表明:在感染50 PFU(plague formation Units)/孔的VSV后,在(4.3.6,3.2.16)細胞株均未發(fā)現空斑,而(1.4.5)細胞克隆中只出現少量空斑,對照組中出現大量的空斑。在0.001 TCID50 VSV對細胞感染48h后病毒產量分別是(4.3.6,3.2.16,1.4.5)3.5X10~3和4.8X10~3和5.3X10~5而對照組中病毒產量為3.5X10~6。三株細胞表現出強烈的抗VSV活性(TCID_(50)提高了1000X)。上述的結果表明我們已經獲得了MxA基因穩(wěn)定轉染NIH-3T3細胞株,并對VSV病毒具有很強的抗性活性,為進一步開展MxA基因抗其他病毒的研究提供實驗基礎。
[Abstract]:The eukaryotic expression vector pEGFP-C1 was constructed by constructing human anti-influenza virus resistance protein A gene, and then pEGFP-C1-MxA was transfected into NIH-3T3 cells. Double positive cell clones were screened by G418 resistance and green fluorescence. The mouse anti-#en4# protein serum was prepared in our laboratory. The above cell clones were analyzed by Immunoblot and immunofluorescence / cell immunocytochemistry, and the specific MxA sequence of the above cells was analyzed and identified by RT-PCR technique. The results showed that the NIH-3T3 cells transfected with VSV were screened by G418 resistance and green fluorescent protein, and three double positive cell clones were screened by two rounds of subcloning. The results showed that three double positive cell clones, 1.4.5FP-C1-MxA, and EGFP gene transfection clones, were selected. The specific primers designed by the sequence of the non-homologous region of the MxA gene and the mouse Mxl gene were used in the RT-PCR test. The results of immunoblot analysis showed that the three cells were strongly positive. The results of immunocytochemistry analysis showed that the three cells above 98% showed strong positive. VSV resistance test showed that after 50 PFU(plague formation Units)/ hole VSV infection, no plaque was found in all the three cell lines, but only a small number of plaque appeared in the cell clone. A large number of plaque appeared in the control group. After 48h infection with 0. 001 TCID50 VSV, the virus production was 4. 3. 6%, 3. 2. 16%, 1. 4. 5%, 3. 5 X 10, 3 and 5. 3 X 10, 5, respectively, while the virus production in the control group was 3. 5 X 10, 10, 6. The three cell lines showed strong anti-VSV activity, TCIDS50) and the above results were increased. This indicates that we have obtained MxA gene stably transfected into NIH-3T3 cell line. And the VSV virus has strong resistance activity, which provides the experimental basis for further research on the resistance of MxA gene to other viruses.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【引證文獻】

相關碩士學位論文 前2條

1 呼都特;蒙古馬不同類群MxA基因的部分外顯子多態(tài)性分析及MxA基因真核載體的構建[D];內蒙古農業(yè)大學;2010年

2 田智泉;雞Mx基因表達及其抗體血清制備的研究[D];揚州大學;2010年

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本文編號：1638734