人抗病毒基因-MxA转染细胞株-NIH-3T3的建立及抗VSV病毒效应

发布时间：2018-03-20 10:44

本文选题：MxA　切入点：NIH-3T3细胞　出处：《苏州大学》2008年硕士论文　论文类型：学位论文

【摘要】： 构建人的抗流感病毒基因-MxA(Myxovirus resistance protein A)克隆真核表达载体pEGFP-C1,再将pEGFP-C1-MxA转染NIH-3T3细胞,通过G418抗性结合绿色荧光筛选出双阳性细胞克隆,用本室制备的鼠抗MxA蛋白血清,对上述的细胞克隆进行Immunoblot分析和细胞免疫荧光/细胞免疫化学分析,并用RT-PCR技术对上述细胞中的MxA特异性序列进行分析鉴定,再用标准VSV病毒株攻击进行抗病毒效应试验。结果显示:pEGFP-C1-MxA转染的NIH-3T3细胞经G418抗性和绿色荧光蛋白筛选并经二轮亚克隆筛选了3个双阳性细胞克隆(1.4.5,4.3.6,3.2.16)和EGFP基因转染克隆;以MxA基因与鼠Mxl基因非同源区的序列设计的特异的引物进行RT-PCR试验三株均显阳性,Immunoblot分析三株细胞表现强阳性,免疫细胞化学分析显示上述三株细胞有大于98%的细胞表现为强阳性。VSV抗性试验表明:在感染50 PFU(plague formation Units)/孔的VSV后,在(4.3.6,3.2.16)细胞株均未发现空斑,而(1.4.5)细胞克隆中只出现少量空斑,对照组中出现大量的空斑。在0.001 TCID50 VSV对细胞感染48h后病毒产量分别是(4.3.6,3.2.16,1.4.5)3.5X10~3和4.8X10~3和5.3X10~5而对照组中病毒产量为3.5X10~6。三株细胞表现出强烈的抗VSV活性(TCID_(50)提高了1000X)。上述的结果表明我们已经获得了MxA基因稳定转染NIH-3T3细胞株,并对VSV病毒具有很强的抗性活性,为进一步开展MxA基因抗其他病毒的研究提供实验基础。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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