【摘要】：目的 設(shè)計(jì)特異性結(jié)合乙肝病毒核心啟動(dòng)子的人工轉(zhuǎn)錄因子并觀察其對(duì)乙肝病毒轉(zhuǎn)錄的抑制作用。 方法 1、利用Zine finger tools設(shè)計(jì)特異性靶向HBV核心啟動(dòng)子的鋅指蛋白，獲得鋅指蛋白的靶序列及氨基酸序列，并對(duì)該蛋白的理化性質(zhì)和結(jié)構(gòu)特征進(jìn)行分析。 2、將ZFP氨基酸序列逆向翻譯成核苷酸序列并進(jìn)行優(yōu)化后人工合成。用帶有核定位信號(hào)(NLS)的引物擴(kuò)增ZFP片段并將其克隆至真核表達(dá)質(zhì)粒pcDNA3.1(+)，構(gòu)建pcDNA3.1(+)-ZFP，然后以此為基礎(chǔ)插入帶有Flag tag的KRAB序列，構(gòu)建人工轉(zhuǎn)錄因子的真核表達(dá)質(zhì)粒pcDNA3.1(+)-ATF。 3、脂質(zhì)體轉(zhuǎn)染重組質(zhì)粒pcDNA3.1(+)-ATF到COS-7細(xì)胞，應(yīng)用RT-PCR和Western Blot分別從mRNA水平和蛋白質(zhì)水平檢測(cè)ATF在真核細(xì)胞中的表達(dá)情況。 4、用脂質(zhì)體將重組質(zhì)粒pcDNA3.1(+)-ATF或pcDNA3.1(+)分別轉(zhuǎn)染HepG2.2.15細(xì)胞，ELISA和FQ-PCR檢測(cè)上清液中HBeAg和HBVDNA含量，RT-PCR檢測(cè)細(xì)胞內(nèi)HBV mRNA表達(dá)，免疫細(xì)胞化學(xué)檢測(cè)細(xì)胞內(nèi)核心抗原的表達(dá)。 結(jié)果 1、鋅指蛋白在HBV核心啟動(dòng)子上的靶序列為：5'-AATGTCAACGACCGACCT-3' 2、鋅指蛋白的氨基酸序列為：LEPGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYKCPECGKSFSQSGHLTEHQRTHTGEKPYKCPECGKSFSDPGNLVRHQRTHTGEKPYKCPECGKSFSDSGNLRVHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSTTGNLTVHQRTHTGKKTS 3、ZFP結(jié)構(gòu)特征分析為：等電點(diǎn)9.20，分子量19.69，穩(wěn)定指數(shù)為31.41，顯示為穩(wěn)定結(jié)構(gòu)。二級(jí)結(jié)構(gòu)預(yù)測(cè)提示該蛋白含有6段Ｃ2Ｈ2型鋅指單元序列，分別位于其氨基酸殘基序列的第8~30、36~58、64~86、92~114、120~142及148~170位。同源建模也提示其含有6個(gè)連續(xù)的鋅指結(jié)構(gòu)。StructureAssessment對(duì)建模結(jié)果進(jìn)行評(píng)估，結(jié)果顯示空間結(jié)構(gòu)穩(wěn)定。 4、pcDNA3.1(+)-ATF轉(zhuǎn)染COS-7細(xì)胞后，經(jīng)RT-PCR及Western Blot檢測(cè)，，ATF mRNA和融合有Flag tag的ATF蛋白表達(dá)明顯增加，而轉(zhuǎn)染pcDNA3.1(+)的細(xì)胞內(nèi)則沒(méi)有相應(yīng)的表達(dá)，說(shuō)明ATF在真核細(xì)胞內(nèi)是能夠正常表達(dá)的。 5、ATF重組質(zhì)粒轉(zhuǎn)染入HepG2.2.15細(xì)胞后，細(xì)胞上清液中的HBVDNA和HBeAg含量明顯減少，細(xì)胞內(nèi)的HBV mRNA及HBcAg也顯著降低，說(shuō)明ATF能夠有效抑制HBV的轉(zhuǎn)錄。 結(jié)論 利用生物信息學(xué)方法設(shè)計(jì)了能特異性結(jié)合HBV核心啟動(dòng)子的鋅指蛋白，并以此為基礎(chǔ)構(gòu)建了完整的人工轉(zhuǎn)錄因子。實(shí)驗(yàn)證明設(shè)計(jì)的人工轉(zhuǎn)錄因子能夠在真核細(xì)胞內(nèi)正常表達(dá)，并對(duì)細(xì)胞模型中的HBV轉(zhuǎn)錄起抑制作用。
[Abstract]:Objective to design the artificial transcription factors specifically binding to HBV core promoter and to observe its inhibitory effect on HBV transcription. Methods 1. Zinc finger protein targeting the core promoter of HBV was designed by Zine finger tools. The target sequence and amino acid sequence of zinc finger protein were obtained, and the physical and chemical properties and structural characteristics of the protein were analyzed. 2. The amino acid sequence of ZFP was converse translated into nucleotide sequence and optimized for artificial synthesis. The ZFP fragment was amplified with a primer with nuclear localization signal (NLS) and cloned into eukaryotic expression plasmid pcDNA3.1 (), to construct pcDNA3.1 ()-ZFP, and then inserted into the KRAB sequence with Flag tag. Construction of eukaryotic expression plasmid pcDNA3.1 ()-ATF. of artificial transcription factors 3. The recombinant plasmid pcDNA3.1 ()-ATF was transfected into COS-7 cells by liposome. The expression of ATF in eukaryotic cells was detected by RT-PCR and Western Blot from mRNA level and protein level, respectively. 4Recombinant plasmids pcDNA3.1 ()-ATF or pcDNA3.1 () were transfected into HepG2.2.15 cells by liposome, HBeAg and HBVDNA contents in supernatant were detected by ELISA and FQ-PCR, HBV mRNA expression was detected by RT-PCR. The expression of core antigen was detected by immunocytochemistry. Results 1. The target sequence of zinc finger protein on the core promoter of HBV was 5- AATGTCAACGACCGACCT-3'2, and the amino acid sequence of zinc finger protein was as follows: isoelectric point 9.20, molecular weight 19.69, and amino acid sequence of zinc finger protein as follows: isoelectric point 9.20, molecular weight 19.69, amino acid sequence of zinc finger protein as follows. The stability index is 31.41, showing a stable structure. The prediction of the secondary structure suggested that the protein contained six C2H2 zinc finger units, located at position 830 / 36 / 586 / 864 / 866 / 92C / 114120 / 142 and at position 148170of the amino acid residues, respectively. The homologous modeling also indicates that it contains six consecutive zinc finger structures. StructureAssessment evaluates the modeling results and the results show that the spatial structure is stable. 4After transfection of COS-7 cells with pcDNA3.1 ()-ATF, the expression of, ATF mRNA and ATF fused with Flag tag was detected by RT-PCR and Western Blot, but there was no corresponding expression in pcDNA3.1 () transfected cells. These results suggest that ATF can be expressed normally in eukaryotic cells. After transfection into HepG2.2.15 cells, the contents of HBVDNA and HBeAg in the supernatant were significantly decreased, and the HBV mRNA and HBcAg in the cells were also significantly decreased, which indicated that ATF could effectively inhibit the transcription of HBV. Conclusion the zinc finger protein which can specifically bind to the core promoter of HBV was designed by bioinformatics, and a complete artificial transcription factor was constructed based on it. The results show that the designed artificial transcription factors can express normally in eukaryotic cells and inhibit the transcription of HBV in the cell model.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R373

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