本文選題：MxA　切入點(diǎn)：NIH-3T3細(xì)胞　出處：《蘇州大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 構(gòu)建人的抗流感病毒基因-MxA(Myxovirus resistance protein A)克隆真核表達(dá)載體pEGFP-C1,再將pEGFP-C1-MxA轉(zhuǎn)染NIH-3T3細(xì)胞,通過(guò)G418抗性結(jié)合綠色熒光篩選出雙陽(yáng)性細(xì)胞克隆,用本室制備的鼠抗MxA蛋白血清,對(duì)上述的細(xì)胞克隆進(jìn)行Immunoblot分析和細(xì)胞免疫熒光/細(xì)胞免疫化學(xué)分析,并用RT-PCR技術(shù)對(duì)上述細(xì)胞中的MxA特異性序列進(jìn)行分析鑒定,再用標(biāo)準(zhǔn)VSV病毒株攻擊進(jìn)行抗病毒效應(yīng)試驗(yàn)。結(jié)果顯示:pEGFP-C1-MxA轉(zhuǎn)染的NIH-3T3細(xì)胞經(jīng)G418抗性和綠色熒光蛋白篩選并經(jīng)二輪亞克隆篩選了3個(gè)雙陽(yáng)性細(xì)胞克隆(1.4.5,4.3.6,3.2.16)和EGFP基因轉(zhuǎn)染克隆;以MxA基因與鼠Mxl基因非同源區(qū)的序列設(shè)計(jì)的特異的引物進(jìn)行RT-PCR試驗(yàn)三株均顯陽(yáng)性,Immunoblot分析三株細(xì)胞表現(xiàn)強(qiáng)陽(yáng)性,免疫細(xì)胞化學(xué)分析顯示上述三株細(xì)胞有大于98%的細(xì)胞表現(xiàn)為強(qiáng)陽(yáng)性。VSV抗性試驗(yàn)表明:在感染50 PFU(plague formation Units)/孔的VSV后,在(4.3.6,3.2.16)細(xì)胞株均未發(fā)現(xiàn)空斑,而(1.4.5)細(xì)胞克隆中只出現(xiàn)少量空斑,對(duì)照組中出現(xiàn)大量的空斑。在0.001 TCID50 VSV對(duì)細(xì)胞感染48h后病毒產(chǎn)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(duì)照組中病毒產(chǎn)量為3.5X10~6。三株細(xì)胞表現(xiàn)出強(qiáng)烈的抗VSV活性(TCID_(50)提高了1000X)。上述的結(jié)果表明我們已經(jīng)獲得了MxA基因穩(wěn)定轉(zhuǎn)染NIH-3T3細(xì)胞株,并對(duì)VSV病毒具有很強(qiáng)的抗性活性,為進(jìn)一步開(kāi)展MxA基因抗其他病毒的研究提供實(shí)驗(yàn)基礎(chǔ)。
[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(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)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(gòu)建[D];內(nèi)蒙古農(nóng)業(yè)大學(xué);2010年

2 田智泉;雞Mx基因表達(dá)及其抗體血清制備的研究[D];揚(yáng)州大學(xué);2010年

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本文編號(hào)：1638734