人乳头瘤病毒16型E6蛋白真核表达及对巨噬细胞分泌功能与凋亡的影响

发布时间：2018-04-04 16:22

本文选题：人乳头瘤病毒16型(HPV　切入点：16)　出处：《南华大学》2006年硕士论文

【摘要】：目的：构建人乳头瘤病毒16型(HPV 16)E6蛋白真核表达载体pcDNA3.1(-)／E6，研究E6蛋白瞬时高表达对LPS诱导的THP-1巨噬细胞细胞因子分泌及地塞米松诱导的巨噬细胞凋亡的影响，为进一步研究E6蛋白在HPV16致癌机理中的作用提供一定的实验依据。方法：用Primer5.0引物设计软件设计引物，聚合酶链反应(PCR)扩增HPV16 E6基因。将PCR产物纯化后与pUCm-T载体连接，经蓝白斑筛选、双酶切鉴定及测序分析后，亚克隆至pcDNA3.1(-)真核表达载体中；重组质粒经双酶切鉴定后转染THP-1巨噬细胞，SDS-PAGE与Western-blotting鉴定E6蛋白在THP-1巨噬细胞中的表达；将pcDNA3.1(-)／E6转染THP-1巨噬细胞，转染后24h加入终浓度为100ng／mL的LPS诱导，分别在诱导后12h、24h、48h收集培养上清用ELISA法检测细胞培养上清中TNF-α、IL-1β的含量，并用显微计数法观察E6蛋白瞬时高表达对地塞米松诱导的巨噬细胞凋亡的影响。利用统计软件SPSS12.0对实验数据作统计分析。结果： (1)PCR产物克隆至pUCm-T载体上后，双酶切及测序结果表明所克隆的目的片段与GenBank上公布的HPV16 E6基因序列完全一致，将该目的片段亚克隆至真核载体pcDNA3.1(-)上，所构建的真核表达载体pcDNA3.1(-)／E6转染THP-1巨噬细胞，，SDS-PAGE与Western-blotting结果显示pcDNA3.1(-)／E6可以在THP-1巨噬细胞中表达分子量约为18KD的E6蛋白。 (2)pcDNA3.1(-)／E6+LPS组培养上清中TNF-α、IL-1 β的量明显低于巨噬细胞+LPS
[Abstract]:Aim: to construct the eukaryotic expression vector pcDNA3.1 16)E6 / E6 of human papillomavirus type 16 (HPV16) and to investigate the effect of transient overexpression of E6 protein on THP-1 macrophage cytokine secretion induced by LPS and dexamethasone induced macrophage apoptosis.To further study the role of E6 protein in the carcinogenesis mechanism of HPV16.Methods: the primers were designed with Primer5.0 primer design software and HPV16 E6 gene was amplified by polymerase chain reaction (PCR).After purification, PCR product was ligated with pUCm-T vector, screened by blue and white spot, identified by double enzyme digestion and sequenced, then subcloned into eukaryotic expression vector pcDNA3.1 ~ (-).The expression of E6 protein in THP-1 macrophages was identified by SDS-PAGE and Western-blotting, and the expression of E6 protein in THP-1 macrophages was identified by SDS-PAGE and Western-blotting. After transfection of pcDNA3.1(-)/E6 into THP-1 macrophages, the final concentration of 100ng/mL was added to LPS at 24 h after transfection, and the expression of E6 protein in THP-1 macrophages was induced by SDS-PAGE and Western-blotting.The supernatants were collected and collected at 12h ~ 24h and 48h after induction. The content of TNF- 伪伪 -IL-1 尾 in the supernatants was detected by ELISA method, and the effect of transient overexpression of E6 protein on the apoptosis of macrophages induced by dexamethasone was observed by microscopic counting method.Statistical software SPSS12.0 is used to make statistical analysis of experimental data.Results:After the product was cloned into pUCm-T vector, the result of double enzyme digestion and sequencing showed that the cloned target fragment was identical with the sequence of HPV16 E6 gene published on GenBank, and the target fragment was subcloned into eukaryotic vector pcDNA3.1 ~ (-).The constructed eukaryotic expression vector pcDNA3.1(-)/E6 was transfected into THP-1 macrophages by SDS-PAGE and Western-blotting. The results showed that pcDNA3.1(-)/E6 could express E6 protein with molecular weight of 18KD in THP-1 macrophages.The content of TNF- 伪 and IL-1 尾 in the supernatant of pcDNA3.1% E6 LPS group was significantly lower than that of macrophage LPS.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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