乙型肝炎病毒X蛋白与hDaxx相互作用及对HepG2细胞凋亡的影响

发布时间：2018-05-06 11:22

  本文选题：乙型肝炎病毒 + X蛋白　； 参考：《南华大学》2007年硕士论文

【摘要】： 目的:应用酵母双杂交法检测乙型肝炎病毒(HBV)X蛋白(HBx)与hDaxx的相互作用,并初步探讨其相互作用后对细胞凋亡的影响,为进一步研究HBx在HBV慢性感染致癌机制中的作用提供一定的实验依据。 方法: (1)构建酵母菌真核表达载体pGADT7-HBx:根据已知的HBV X基因序列,采用Primer Premier5.0软件设计一对引物,并分别引入EcoR I与Xho I酶切位点,PCR扩增X基因;用EcoRI和Xho I分别双酶切PCR产物和质粒pGADT7,分别纯化回收酶切产物,T4连接酶连接后,转化大肠杆菌DH5α,筛选阳性克隆。对重组质粒进行酶切及测序鉴定。 (2)构建酵母菌真核表达载体pGBKT7-hDaxx:用EcoRI和SalI分别双酶切质粒pGBDU-C1/hDaxx和pGBKT7,分别纯化回收酶切产物,T4连接酶连接后,转化大肠杆菌DH5α,筛选到阳性克隆后,对重组质粒行酶切鉴定。 (3)酵母双杂交检测HBx与hDaxx的相互作用:将酵母菌真核表达载体及酵母菌阴性阳性对照质粒分4组转化酵母菌AH109,A组为pGADT7和pGBKT7、B组为pGADT7-HBx和pGBKT7-hDaxx、C组为pGADT7-T和pGBKT7-Lam、D组为pGADT7-T和pGBKT7-p53,将转化菌落接种于SD/-Trp-Leu(二缺)固体平板,30℃培养2～4d。将单个菌落接种于SD/-Trp-Leu-His(三缺)和SD/-Trp-Leu-His-Ade(四缺)平板,30℃培养2～4d。裂解酵母菌,提取蛋白,经SDS-PAGE、Western blot检测hDaxx和HBx在酵母中的表达。 (4)流式细胞术检测细胞凋亡:将HBV X基因稳定转染入HepG2细胞(即HepG2X),再瞬时转染15μg、30μg和45μg pcDAN3.1-hDaxx,以pcDNA3.1(+)转染HepG2X组和HepG2X组分别作为对照。用10mmol/L的5-氟尿嘧啶(5-FU)处理36h后分别收集各组细胞,经75%的乙醇4℃固定过夜,PI染色后,用流式细胞仪检测各组细胞的凋亡率。用统计软件SPSS13.0对实验数据进行LSD-t检验分析。 结果: (1) PCR扩增HBV X基因片段(465bp),将X基因克隆至pGADT7载体上,经双酶切及测序分析,所克隆的目的片段与已知的X基因序列及GeneBank上公布的HBV X基因(Pubmed NC_U95551)序列完全一致。 (2)将pGBDU-C1/hDaxx上的hDaxx基因片段亚克隆至pGBKT7载体上,经双酶切分析,得约2.2kb大小的hDaxx基因片段,与预期值大小一致。 (3)分4组转化的酵母菌AH109在二缺陷平板上均可长出白色菌落,但仅有B、D两组菌可以在三缺和四缺平板上生长,选取B组四缺陷平板上单个菌落,提取酵母蛋白, Western blot检测到hDaxx和HBx在同一株酵母菌株中均有表达。 (4)将X基因稳定转染入HepG2细胞,Western blot检测到HBx在HepG2X细胞中的表达。经流式细胞术检测,HepG2细胞用5-FU处理后凋亡率明显高于未经5-FU处理的细胞组,结果具有显著性差异(P0.01);HepG2X组细胞凋亡率明显低于空细胞组,结果具有显著性差异(P0.01);而转染pcDAN3.1-hDaxx后,细胞凋亡率进一步降低,但与转染入的pcDAN3.1-hDaxx量没有剂量依赖关系。 结论: (1)成功地构建了pGBKT7-hDaxx和pGADT7-HBx酵母菌真核表达载体,并可在酵母菌AH109中表达。 (2) HBx与hDaxx在酵母细胞内存在相互作用。 (3)构建了稳定表达HBx的细胞株HepG2X。 (4) HBx可抑制5-FU诱导的HepG2细胞凋亡,而hDaxx的高表达能提高HBx的抑制作用,使细胞凋亡率进一步降低。
[Abstract]:Objective: to detect the interaction between hepatitis B virus (HBV) X protein (HBx) and hDaxx by yeast two hybrid method, and to explore the effect of its interaction on cell apoptosis, so as to provide some experimental evidence for further study of the role of HBx in the pathogenesis of HBV chronic infection.
Method:
(1) to construct the eukaryotic expression vector pGADT7-HBx:, a pair of primers were designed based on the known HBV X gene, and Primer Premier5.0 software was used to design a pair of primers, and EcoR I and Xho I enzyme cut sites were introduced, and X gene was amplified by PCR. Escherichia coli DH5 alpha was screened and positive clones were screened. The recombinant plasmid was identified by restriction enzyme digestion and sequencing.
(2) construction of yeast eukaryotic expression vector pGBKT7-hDaxx: with EcoRI and SalI double enzyme cut plasmids pGBDU-C1/hDaxx and pGBKT7 respectively, purification and recovery of the enzyme cut products, T4 ligase connection, the transformation of Escherichia coli DH5 alpha, after screening the positive clones, the recombinant plasmid was identified by enzyme digestion.
(3) the interaction between HBx and hDaxx was detected by yeast two hybrid: the yeast eukaryotic expression vector and the yeast negative positive control plasmid were divided into 4 groups of yeast AH109, A group pGADT7 and pGBKT7, B group pGADT7-HBx and pGBKT7-hDaxx, C group pGADT7-T and pGBKT7-Lam. In the solid plate, a single colony was inoculated to SD/-Trp-Leu-His (three deficient) and SD/-Trp-Leu-His-Ade (four deficient) plate at 30 C for 2 ~ 4d., and 2 ~ 4d. lysis yeast was cultured at 30 C, and protein was extracted. The expression of hDaxx and HBx in yeast was detected by SDS-PAGE and Western blot.
(4) flow cytometry was used to detect apoptosis: transfection of HBV X gene into HepG2 cells (HepG2X), then transiently transfected 15 mu g, 30 mu g and 45 mu g pcDAN3.1-hDaxx. The cells were treated with pcDNA3.1 (+) transfected HepG2X group and HepG2X group respectively. The cells were collected with 10mmol/L's fluorouracil, respectively, after 75% ethanol at 4 centigrade. At night, after PI staining, the apoptosis rate of each group was detected by flow cytometry. The data were analyzed by LSD-t with statistical software SPSS13.0.
Result:
(1) the HBV X gene fragment (465bp) was amplified by PCR, and the X gene was cloned to the pGADT7 vector. The target fragment was completely identical with the known sequence of X gene and the HBV X gene (Pubmed NC_U95551) published on GeneBank by double enzyme digestion and sequencing.
(2) the hDaxx gene fragment on pGBDU-C1/hDaxx was subcloned to pGBKT7 vector, and the hDaxx gene fragment of about 2.2Kb size was obtained by double enzyme cutting analysis, which was in accordance with the expected value.
(3) the yeast AH109 transformed by 4 groups could grow white colonies on two defective plates, but only B, D two could grow on three and four vacant plates, selected a single colony on the B group four defect plate and extracted the yeast protein, and Western blot detected that hDaxx and HBx were expressed in the same strain of yeast strain.
(4) the X gene was transfected into HepG2 cells steadily and the expression of HBx in HepG2X cells was detected by Western blot. The apoptosis rate of HepG2 cells treated with 5-FU was significantly higher than that of the group without 5-FU (P0.01). The apoptotic rate in HepG2X group was significantly lower than that in the empty cell group, and the result was significant. Difference (P0.01), and after transfection of pcDAN3.1-hDaxx, the rate of apoptosis decreased further, but there was no dose-dependent relationship with the amount of pcDAN3.1-hDaxx transfected.
Conclusion:
(1) successfully constructed pGBKT7-hDaxx and pGADT7-HBx yeast eukaryotic expression vectors, and can be expressed in yeast AH109.
(2) there is interaction between HBx and hDaxx in yeast cells.
(3) the cell line HepG2X., which is stable to express HBx, is constructed.
(4) HBx can inhibit the apoptosis of 5-FU induced HepG2 cells, while the high expression of hDaxx can enhance the inhibitory effect of HBx and further reduce the apoptosis rate.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

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