乙型肝炎病毒X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：2018-05-09 00:35

本文选题：乙型肝炎病毒 + X基因　；参考：《南华大学》2006年硕士论文

【摘要】：目的：将乙型肝炎病毒(Hepatitis B virus，HBV)X蛋白真核表达载体pcDNA3.1(+)-HBx转染巨噬细胞，观察X蛋白在细胞中的表达及其对巨噬细胞分泌细胞因子及凋亡的影响，为进一步研究HBV的致病机制奠定实验基础。方法：用Primer 5.0软件分析HBV X基因序列，设计相应特异性引物，聚合酶链反应(PCR)扩增465bp的目的基因片段，将其插入pcDNA3.1(+)载体，通过双酶切分析及测序鉴定筛选阳性重组体；将真核表达载体pcDNA3.1(+)-HBx通过Super Fect~(TM)脂转染试剂转染巨噬细胞，，利用Western-blotting鉴定X蛋白在巨噬细胞中的表达；细胞转染质粒后24h，用LPS刺激巨噬细胞，酶联免疫吸附试验(ELISA)检测pcDNA3.1(+)-HBx转染的巨噬细胞不同时间(12h、24h、48h)培养上清液中细胞因子TNF-α、IL-1β的含量，统计软件SPSS 13.0分析所得数据；通过形态学方法观察巨噬细胞，利用显微计数法测定X蛋白即时高表达时，地塞米松诱导的细胞凋亡并计算凋亡率，用统计软件通过方差分析所得数据。结果： (1)将PCR扩增的465bp大小的HBV X基因片段，连接入pcDNA3.1(+)，双酶切及测序结果表明X基因亚克隆入pcDNA3.1(+)真核表达载体，即得X蛋白真核表达载体pcDNA3.1(+)-HBx。 (2)将真核表达载体pcDNA3.1(+)-HBx转染巨噬细胞，Western-blotting结果显示pcDNA3.1(+)-HBx能在巨噬细胞中表达约17KD的目的蛋白。 (3)ELISA法测定细胞上清液中TNF-α、IL-1β的含量，LPS刺激组及pcDNA3.1(+)组与空白对照组有显著性差异(P＜0.01)，pcDNA3.1(+)-HBx组与LPS刺激组及pcDNA3.1(+)组有显著性差异(P＜0.01)，而LPS刺激组与pcDNA3.1(+)组无显著性差异(P＞0.05)，即LPS可活化巨噬细胞分泌细胞因子，且X蛋白即时高表达可引起LPS诱导的巨噬细胞
[Abstract]:Objective: to investigate the expression of X protein in macrophages and its effect on the secretion of cytokines and apoptosis of macrophages. To lay an experimental foundation for the further study of the pathogenetic mechanism of HBV. Methods: the sequence of HBV X gene was analyzed by Primer 5.0 software. Specific primers were designed. The target gene fragment of 465bp was amplified by polymerase chain reaction (PCR) and inserted into pcDNA3.1 () vector. The positive recombinant was screened by double enzyme digestion and sequencing. The eukaryotic expression vector pcDNA3.1was transfected into macrophages by Super FectCT-TMT lipid transfection reagent. The expression of X protein in macrophages was identified by Western-blotting, and the macrophages were stimulated by LPS at 24 hours after transfection. Enzyme linked immunosorbent assay (Elisa) was used to detect the levels of cytokine TNF- 伪 and IL-1 尾 in the supernatant of macrophages transfected with pcDNA3.1 (24 h ~ 48 h) at different times. The data were analyzed by SPSS 13.0, and the macrophages were observed by morphological method. The apoptosis induced by dexamethasone and the apoptosis rate were measured by microcounting method. The data were obtained by variance analysis with statistical software. Results: The HBV X gene fragment amplified by PCR was ligated into pcDNA3.1. the results of double enzyme digestion and sequencing showed that X gene was subcloned into pcDNA3.1 () eukaryotic expression vector, namely X protein eukaryotic expression vector pcDNA3.1 (-HBx.) The results of Western-blotting analysis showed that pcDNA3.1 (-HBx) could express the target protein of about 17KD in macrophages. Determination of TNF- 伪 LPS 尾 in Cell supernatant by Elisa; there was significant difference between LPS-stimulated group and pcDNA3.1 () group and blank control group (P < 0.01). (P < 0.01). There was significant difference between LPS group and pcDNA3.1group (P < 0.01), but there was no significant difference between LPS group and pcDNA3.1 () group (P > 0.05), that is to say, there was no significant difference between LPS-stimulated group and pcDNA3.1 () group (P > 0.05), that is, the difference was significant (P < 0.01) between LPS group and pcDNA3.1 group (P < 0.01), but there was no significant difference between LPS-stimulated group and pcDNA3.1 group (P > 0.05). LPS activates macrophages to secrete cytokines. And immediate overexpression of X protein could induce macrophages induced by 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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