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  本文選題：miR-122 + 真核表達(dá)載體�。� 參考：《華中科技大學(xué)》2008年碩士論文

【摘要】： 目的 1.構(gòu)建肝臟特異性表達(dá)的微小RNA——miR-122的真核表達(dá)載體; 2.將該重組質(zhì)粒在人肝癌細(xì)胞系中轉(zhuǎn)錄,通過現(xiàn)代分子生物學(xué)技術(shù)鑒定其生物活性。 3.利用該重組質(zhì)粒初步探討miR-122與乙型肝炎病毒復(fù)制之間的關(guān)系。 方法 1.真核表達(dá)載體的構(gòu)建: 應(yīng)用PCR技術(shù),以從人類肝癌細(xì)胞中提取的基因組DNA為模板,獲得miR-122的編碼基因片段。根據(jù)設(shè)計的酶切位點,將其與真核表達(dá)載體pSuper同時雙酶切,然后經(jīng)T4連接酶連接,篩選得到穩(wěn)定的克隆,命名為pHsa-m122。 2.鑒定重組體的生物活性: 以美國斯坦福大學(xué)醫(yī)學(xué)院微生物與免疫教研室Peter Sarnow教授饋贈的熒光報告質(zhì)粒GFP-122 sensor(GFP-122si)為報告質(zhì)粒、GFP-124 sensor (GFP-124si)為無關(guān)對照質(zhì)粒,該報告質(zhì)粒GFP-122si的轉(zhuǎn)錄可被成熟的miR-122干擾,使綠色熒光蛋白的表達(dá)量下降,同樣GFP-124si的轉(zhuǎn)錄也可被成熟的miR-124干擾,而不受成熟的miR-122干擾。因而將pHsa-m122與報告質(zhì)粒GFP-122si共同轉(zhuǎn)染人肝癌細(xì)胞系HepG2,以pHsa-m122與參照質(zhì)粒GFP-124si共轉(zhuǎn)染人肝癌細(xì)胞系HepG2為對照,分別用倒置熒光顯微鏡觀察綠色熒光蛋白的表達(dá)、流式細(xì)胞儀檢測細(xì)胞的平均熒光強(qiáng)度、Western blot檢測綠色熒光蛋白的表達(dá)量這三種方式來檢測GFP的表達(dá)變化,通過GFP蛋白表達(dá)量的下降來反映成熟的miR-122的表達(dá)情況。 3.初步探索miR-122與HBV復(fù)制之間的關(guān)系: 通過體外真核轉(zhuǎn)染確定質(zhì)粒pHsa-m122的有效性后,將pHsa-m122轉(zhuǎn)染入人肝癌細(xì)胞系HepG2.2.15細(xì)胞,經(jīng)過24小時后,用ELISA的方法來檢測細(xì)胞分泌上清中HBsAg、HBeAg的表達(dá)量。 結(jié)果 1.真核表達(dá)載體的構(gòu)建: pHsa-m122經(jīng)PCR鑒定,能夠獲得預(yù)期大小的目的片段; pHsa-m122經(jīng)雙酶切鑒定,可獲得預(yù)期大小片段; pHsa-m122經(jīng)Invitrogen公司測序證實,miR-122的編碼基因片段已經(jīng)正確插入pSuper載體中。 2.載體的生物活性鑒定: 熒光、流式、Western blot的結(jié)果均表明:pHsa-m122與報告質(zhì)粒GFP-122si共同轉(zhuǎn)染人肝癌細(xì)胞系HepG2,能夠有效抑制綠色熒光蛋白的表達(dá)。因此證實pHsa-m122可轉(zhuǎn)錄出具有生物活性的成熟的miR-122; 3.載體的初步應(yīng)用: 將pHsa-m122轉(zhuǎn)染入人肝癌細(xì)胞系HepG2.2.15后,檢測到細(xì)胞分泌上清中的HBsAg、HBeAg的表達(dá)量升高。 結(jié)論 1. miR-122的前體序列成功地克隆到真核表達(dá)載體pSuper上,miR-122的真核表達(dá)載體pHsa-m122克隆成功; 2. pHsa-m122可以表達(dá)出具有生物活性的成熟的miR-122; 3.在對miR-122與HBV復(fù)制關(guān)系的初步探索中,我們的實驗結(jié)果提示miR-122可能與乙型肝炎病毒的復(fù)制有一定關(guān)系。miR-122真核表達(dá)載體pHsa-m122的成功構(gòu)建,為進(jìn)一步研究miR-122在肝炎病毒復(fù)制及肝癌形成中的作用奠定了實驗基礎(chǔ)。 本課題的創(chuàng)新點 1.在國內(nèi)首次通過檢測報告質(zhì)粒表達(dá)綠色熒光蛋白的量的方法來檢測微小RNA真核表達(dá)載體的生物活性,并運(yùn)用熒光、流式、Western blot的方法來鑒定出微小RNA真核表達(dá)載體的生物活性。 2.在國內(nèi)首次將微小RNA的真核表達(dá)載體運(yùn)用到對乙型肝炎病毒復(fù)制的研究探索中。
[Abstract]:Purpose



1 . constructing a eukaryotic expression vector of a small RNA _ miR - 122 which is specifically expressed in the liver ;



2 . The recombinant plasmid is transcribed in human liver cancer cell line , and its biological activity is identified by modern molecular biology technique .



3 . The relationship between miR - 122 and replication of hepatitis B virus was investigated by using the recombinant plasmid .



method



1 . Construction of eukaryotic expression vector :



The gene fragment of miR - 122 was obtained from genomic DNA extracted from human liver cancer cells by using PCR technique . According to the designed enzyme cleavage site , it was digested with double enzyme at the same time as eukaryotic expression vector pSuper , then ligated with T4 ligase , screened to obtain stable clone , named pHsa - m122 .



2 . Identification of the biological activity of the recombinant :



The expression of green fluorescent protein was detected by an inverted fluorescence microscope , and the expression of green fluorescent protein was detected by Western blot . The expression of GFP - 122 was detected by Western blot .



3 . Initial exploration of the relationship between miR - 122 and HBV replication :



pHsa - m122 was transfected into HepG2.2 . 15 cell line HepG2.2 . 15 . After 24 hours , the expression of HBsAg and HBeAg was detected by ELISA .



Results



1 . Construction of eukaryotic expression vector :



pHsa - m122 was identified by PCR to obtain the desired fragment of the desired size ; pHsa - m122 was identified by double enzyme digestion to obtain the desired size fragment ; pHsa - m122 was confirmed by sequencing by Invitrogen Corporation , and the encoded gene fragment of miR - 122 was correctly inserted into the pSuper vector .



2 . Identification of the biological activity of the carrier :



The results of fluorescence , flow and Western blot showed that pHsa - m122 co - transfected human liver cancer cell line HepG2 with reporter plasmid GFP - 122 si , which can effectively inhibit the expression of green fluorescent protein . Therefore , it was confirmed that pHsa - m122 could transcribe mature miR - 122 ; 3 . vector preliminary application :



After transfection of pHsa - m122 into human hepatoma cell line HepG2.2 . 15 , the expression of HBsAg and HBeAg in the supernatant was detected .



Conclusion



1 . The precursor sequence of miR - 122 is successfully cloned into the eukaryotic expression vector pSuper , and the eukaryotic expression vector pHsa - m122 of miR - 122 is cloned successfully ;



2 . pHsa - m122 can express the mature miR - 122 with biological activity ;



3 . In the preliminary exploration of the relationship between miR - 122 and HBV replication , our experimental results suggest that miR - 122 may be associated with replication of hepatitis B virus . The successful construction of miR - 122 eukaryotic expression vector pHsa - m122 provides an experimental basis for further research on the role of miR - 122 in the replication of hepatitis virus and the formation of liver cancer .



Innovation Points of the Project



1 . The biological activity of the eukaryotic expression vector was first detected by detecting the amount of green fluorescent protein expressed in the reporter plasmid at home for the first time , and the bioactivity of the eukaryotic expression vector was identified by fluorescence , flow and Western blot .



2 . For the first time , the eukaryotic expression vector of microRNA was applied to the study of hepatitis B virus replication .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R346

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本文編號：2013198