本文選題：乙型肝炎病毒 + X基因��； 參考：《南華大學》2006年碩士論文

【摘要】：目的：將乙型肝炎病毒(Hepatitis B virus，HBV)X蛋白真核表達載體pcDNA3.1(+)-HBx轉(zhuǎn)染巨噬細胞，觀察X蛋白在細胞中的表達及其對巨噬細胞分泌細胞因子及凋亡的影響，為進一步研究HBV的致病機制奠定實驗基礎(chǔ)。 方法：用Primer 5.0軟件分析HBV X基因序列，設(shè)計相應特異性引物，聚合酶鏈反應(PCR)擴增465bp的目的基因片段，將其插入pcDNA3.1(+)載體，通過雙酶切分析及測序鑒定篩選陽性重組體；將真核表達載體pcDNA3.1(+)-HBx通過Super Fect~(TM)脂轉(zhuǎn)染試劑轉(zhuǎn)染巨噬細胞，，利用Western-blotting鑒定X蛋白在巨噬細胞中的表達；細胞轉(zhuǎn)染質(zhì)粒后24h，用LPS刺激巨噬細胞，酶聯(lián)免疫吸附試驗(ELISA)檢測pcDNA3.1(+)-HBx轉(zhuǎn)染的巨噬細胞不同時間(12h、24h、48h)培養(yǎng)上清液中細胞因子TNF-α、IL-1β的含量，統(tǒng)計軟件SPSS 13.0分析所得數(shù)據(jù)；通過形態(tài)學方法觀察巨噬細胞，利用顯微計數(shù)法測定X蛋白即時高表達時，地塞米松誘導的細胞凋亡并計算凋亡率，用統(tǒng)計軟件通過方差分析所得數(shù)據(jù)。 結(jié)果： (1)將PCR擴增的465bp大小的HBV X基因片段，連接入pcDNA3.1(+)，雙酶切及測序結(jié)果表明X基因亞克隆入pcDNA3.1(+)真核表達載體，即得X蛋白真核表達載體pcDNA3.1(+)-HBx。 (2)將真核表達載體pcDNA3.1(+)-HBx轉(zhuǎn)染巨噬細胞，Western-blotting結(jié)果顯示pcDNA3.1(+)-HBx能在巨噬細胞中表達約17KD的目的蛋白。 (3)ELISA法測定細胞上清液中TNF-α、IL-1β的含量，LPS刺激組及pcDNA3.1(+)組與空白對照組有顯著性差異(P＜0.01)，pcDNA3.1(+)-HBx組與LPS刺激組及pcDNA3.1(+)組有顯著性差異(P＜0.01)，而LPS刺激組與pcDNA3.1(+)組無顯著性差異(P＞0.05)，即LPS可活化巨噬細胞分泌細胞因子，且X蛋白即時高表達可引起LPS誘導的巨噬細胞
[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- 偽 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- 偽 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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前1條

1 陳金軍;乙型肝炎病毒X基因變異與臨床[J];國外醫(yī)學.病毒學分冊;1998年01期

本文編號：1863809