本文選題：乙型肝炎病毒 + X蛋白��； 參考：《南華大學》2007年碩士論文

【摘要】： 目的:應用酵母雙雜交法檢測乙型肝炎病毒(HBV)X蛋白(HBx)與hDaxx的相互作用,并初步探討其相互作用后對細胞凋亡的影響,為進一步研究HBx在HBV慢性感染致癌機制中的作用提供一定的實驗依據(jù)。 方法: (1)構建酵母菌真核表達載體pGADT7-HBx:根據(jù)已知的HBV X基因序列,采用Primer Premier5.0軟件設計一對引物,并分別引入EcoR I與Xho I酶切位點,PCR擴增X基因;用EcoRI和Xho I分別雙酶切PCR產(chǎn)物和質(zhì)粒pGADT7,分別純化回收酶切產(chǎn)物,T4連接酶連接后,轉(zhuǎn)化大腸桿菌DH5α,篩選陽性克隆。對重組質(zhì)粒進行酶切及測序鑒定。 (2)構建酵母菌真核表達載體pGBKT7-hDaxx:用EcoRI和SalI分別雙酶切質(zhì)粒pGBDU-C1/hDaxx和pGBKT7,分別純化回收酶切產(chǎn)物,T4連接酶連接后,轉(zhuǎn)化大腸桿菌DH5α,篩選到陽性克隆后,對重組質(zhì)粒行酶切鑒定。 (3)酵母雙雜交檢測HBx與hDaxx的相互作用:將酵母菌真核表達載體及酵母菌陰性陽性對照質(zhì)粒分4組轉(zhuǎn)化酵母菌AH109,A組為pGADT7和pGBKT7、B組為pGADT7-HBx和pGBKT7-hDaxx、C組為pGADT7-T和pGBKT7-Lam、D組為pGADT7-T和pGBKT7-p53,將轉(zhuǎn)化菌落接種于SD/-Trp-Leu(二缺)固體平板,30℃培養(yǎng)2～4d。將單個菌落接種于SD/-Trp-Leu-His(三缺)和SD/-Trp-Leu-His-Ade(四缺)平板,30℃培養(yǎng)2～4d。裂解酵母菌,提取蛋白,經(jīng)SDS-PAGE、Western blot檢測hDaxx和HBx在酵母中的表達。 (4)流式細胞術檢測細胞凋亡:將HBV X基因穩(wěn)定轉(zhuǎn)染入HepG2細胞(即HepG2X),再瞬時轉(zhuǎn)染15μg、30μg和45μg pcDAN3.1-hDaxx,以pcDNA3.1(+)轉(zhuǎn)染HepG2X組和HepG2X組分別作為對照。用10mmol/L的5-氟尿嘧啶(5-FU)處理36h后分別收集各組細胞,經(jīng)75%的乙醇4℃固定過夜,PI染色后,用流式細胞儀檢測各組細胞的凋亡率。用統(tǒng)計軟件SPSS13.0對實驗數(shù)據(jù)進行LSD-t檢驗分析。 結(jié)果: (1) PCR擴增HBV X基因片段(465bp),將X基因克隆至pGADT7載體上,經(jīng)雙酶切及測序分析,所克隆的目的片段與已知的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)將pGBDU-C1/hDaxx上的hDaxx基因片段亞克隆至pGBKT7載體上,經(jīng)雙酶切分析,得約2.2kb大小的hDaxx基因片段,與預期值大小一致。 (3)分4組轉(zhuǎn)化的酵母菌AH109在二缺陷平板上均可長出白色菌落,但僅有B、D兩組菌可以在三缺和四缺平板上生長,選取B組四缺陷平板上單個菌落,提取酵母蛋白, Western blot檢測到hDaxx和HBx在同一株酵母菌株中均有表達。 (4)將X基因穩(wěn)定轉(zhuǎn)染入HepG2細胞,Western blot檢測到HBx在HepG2X細胞中的表達。經(jīng)流式細胞術檢測,HepG2細胞用5-FU處理后凋亡率明顯高于未經(jīng)5-FU處理的細胞組,結(jié)果具有顯著性差異(P0.01);HepG2X組細胞凋亡率明顯低于空細胞組,結(jié)果具有顯著性差異(P0.01);而轉(zhuǎn)染pcDAN3.1-hDaxx后,細胞凋亡率進一步降低,但與轉(zhuǎn)染入的pcDAN3.1-hDaxx量沒有劑量依賴關系。 結(jié)論: (1)成功地構建了pGBKT7-hDaxx和pGADT7-HBx酵母菌真核表達載體,并可在酵母菌AH109中表達。 (2) HBx與hDaxx在酵母細胞內(nèi)存在相互作用。 (3)構建了穩(wěn)定表達HBx的細胞株HepG2X。 (4) HBx可抑制5-FU誘導的HepG2細胞凋亡,而hDaxx的高表達能提高HBx的抑制作用,使細胞凋亡率進一步降低。
[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.

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R373

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