本文選題：人工轉(zhuǎn)錄因子 + 鋅指蛋白��； 參考：《重慶醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：設(shè)計出一種新型的人工轉(zhuǎn)錄因子（artificialtranscription factor，ATF），使其能夠特異性結(jié)合于乙型肝炎病毒（hepatitis B virus，HBV） X基因啟動子區(qū)域的靶序列，通過下調(diào)X基因來干擾和抑制HBV DNA的正常的復(fù)制和表達，為乙型肝炎的基因治療提供實驗依據(jù)。 方法：（1）通過生物信息學(xué)工具獲取人工鋅指蛋白（zinc fingerprotein，ZFP）氨基酸序列，經(jīng)過密碼子優(yōu)化后構(gòu)建重組質(zhì)粒pEGFP-N1-ZFP，，將其轉(zhuǎn)染COS-7真核表達細胞，通過觀察綠色熒光蛋白的表達、RT-PCR和Western blot檢測其在真核細胞中的表達；（2）構(gòu)建含有ZFP結(jié)合結(jié)構(gòu)域和KRAB效應(yīng)結(jié)構(gòu)域的ATF，將其轉(zhuǎn)染COS-7真核表達細胞，通過RT-PCR和Western blot檢測其在真核細胞中的表達；（3）將pcDNA3.1(+)-ATF轉(zhuǎn)染HepG2.2.15細胞，通過酶聯(lián)免疫法ELISA、細胞免疫組化、熒光定量方法、RT-PCR等方法檢測其對HBVDNA復(fù)制和表達的抑制作用。結(jié)果：（1）成功構(gòu)建了pEGFP-N1-ZFP重組質(zhì)粒，并驗證其在COS-7細胞中的表達；（2）成功構(gòu)建ATF，并驗證其具有相應(yīng)的生物學(xué)活性；（3）成功驗證了重組質(zhì)粒pcDNA3.1(+)-ATF能夠抑制HepG2.2.15細胞內(nèi)HBV DNA的復(fù)制和表達。 結(jié)論：針對HBV X基因靶序列設(shè)計出來的ATF能夠抑制HepG2.2.15細胞內(nèi)HBV DNA的復(fù)制和表達。
[Abstract]:Objective: to design a novel artificial transcription factor-ATFN to specifically bind to the target sequence of the X promoter region of hepatitis B virus (HBV) gene, and to interfere and inhibit the normal replication and expression of HBV DNA by down-regulating X gene. To provide experimental basis for gene therapy of hepatitis B. Methods the amino acid sequence of artificial zinc finger protein (ZFP) was obtained by bioinformatics. The recombinant plasmid pEGFP-N1-ZFP1 was constructed by codon optimization and transfected into COS-7 eukaryotic expression cells. The expression of green fluorescent protein (GFP) in eukaryotic cells was detected by RT-PCR and Western blot. ATFs containing ZFP binding domain and KRAB effect domain were constructed and transfected into COS-7 eukaryotic expression cells. RT-PCR and Western blot were used to detect its expression in eukaryotic cells. PcDNA3.1 (-ATF) was transfected into HepG2.2.15 cells. The inhibition of HBVDNA replication and expression was detected by Elisa, immunohistochemistry and RT-PCR. Results the recombinant plasmid of pEGFP-N1-ZFP was successfully constructed, and its expression in COS-7 cells was verified. The recombinant plasmid pcDNA3.1 (pDNA3.1) was proved to be able to inhibit the replication and expression of HBV DNA in HepG2.2.15 cells. Conclusion: ATF designed for the target sequence of HBV X gene can inhibit the replication and expression of HBV DNA in HepG2.2.15 cells.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R373

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