【摘要】：世界上大約有3%(1.7億)的人感染了丙型肝炎病毒(HCV)，HCV可引起嚴重的肝臟疾病，如慢性肝炎、肝硬化乃至肝癌。由于缺少針對丙肝的疫苗，且目前的治療方法有一定的局限性，研究新型、高效的抗HCV藥物的重任迫在眉睫。NS2蛋白能夠影響子代病毒顆粒裝配，也能通過干擾宿主細胞的生理過程來促進病毒的復(fù)制。以NS2蛋白作為藥物研究的靶點，具有巨大的潛力。核酸適體是一類新型的識別分子,具有親和力強、選擇性高、穩(wěn)定性好、易于修飾等優(yōu)點，，廣泛用于生物檢測和新藥開發(fā)等方面。在HCV編碼的非結(jié)構(gòu)蛋白中，針對NS2蛋白的藥物尚未見報道。本文以NS2蛋白為靶標，篩選出特異結(jié)合的核酸適體，并研究了適體對病毒生活周期的影響，取得了以下結(jié)果： （1）采用普通PCR方法成功擴增出HCV NS2基因，并將目的基因成功構(gòu)建到pET28b原核表達載體中。 （2）將重組質(zhì)粒pET28b-NS2轉(zhuǎn)到BL21感受態(tài)細胞中進行蛋白大量表達，發(fā)現(xiàn)此蛋白可溶性低。采用超速離心法，并加入膜表面活性劑—月桂酸，可獲得可溶性強的目的蛋白，用鎳柱進一步純化得到蛋白。 （3）以純化的NS2蛋白為靶標，經(jīng)過5輪篩選，得到特異結(jié)合的ssNDAaptamers。從中選擇三條適體（NS2-1、NS2-2、NS2-3）分析發(fā)現(xiàn)，這3種核酸適體能夠特異結(jié)合到NS2蛋白上，不會與對照蛋白HCV NS5B和LacZ發(fā)生非特異性結(jié)合。 （4）用熒光定量PCR和免疫熒光法檢測轉(zhuǎn)染核酸適體后細胞中的病毒復(fù)制和釋放能力變化，發(fā)現(xiàn)核酸適體以一種呈劑量依賴型的方式抑制病毒復(fù)制，此外核酸適體NS2-2、NS2-3還能夠抑制病毒顆粒的釋放。結(jié)果表明針對NS2蛋白的核酸適體能夠抑制病毒的復(fù)制和釋放，有望研究成一種新型的靶向治療HCV的藥物。
[Abstract]:About 3% (170 million) of people in the world are infected with hepatitis C virus (HCV), which can cause serious liver diseases, such as chronic hepatitis, liver cirrhosis and even liver cancer. Due to the lack of a vaccine against hepatitis C and the limitations of current treatment methods, it is urgent to study new and efficient antiviral drugs. NS2 protein can affect the assembly of viral particles in offspring. Virus replication can also be promoted by interfering with the physiological processes of host cells. Using NS2 protein as the target of drug research has great potential. Aptamer is a new class of recognition molecules with high affinity, high selectivity, good stability and easy modification. It is widely used in biological detection and new drug development. Among the non-structural proteins encoded by HCV, drugs targeting NS2 proteins have not been reported. In this paper, NS2 protein was used as the target to screen out the specific binding aptamers, and the effects of aptamers on virus life cycle were studied. The following results were obtained: (1) the HCV NS2 gene was successfully amplified by ordinary PCR method. The target gene was successfully constructed into pET28b prokaryotic expression vector. (2) the recombinant plasmid pET28b-NS2 was transfered into BL21 receptive cells to express the protein in large quantities, and it was found that the protein was low soluble. The soluble target protein was obtained by ultracentrifugation and membrane surfactant-lauric acid. The protein was further purified by nickel column. (3) the purified NS2 protein was used as the target, and the specific binding ssNDAaptamers. was obtained after five rounds of screening. Analysis of three aptamers (NS2-1,NS2-2,NS2-3) showed that the three aptamers could specifically bind to NS2 protein and could not bind to HCV NS5B and LacZ. (4) the ability of virus replication and release after transfection of nucleic acid aptamer was detected by fluorescence quantitative PCR and immunofluorescence method. It was found that the aptamer inhibited virus replication in a dose-dependent manner, and the aptamer NS2-2, inhibited virus replication in a dose-dependent manner. NS2-3 can also inhibit the release of virus particles. The results showed that the aptamer targeting NS2 protein could inhibit the replication and release of the virus, and it was expected to be a novel drug targeting the treatment of HCV.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前5條

1 王成剛;莫志宏;;核酸適體技術(shù)研究進展[J];生物醫(yī)學(xué)工程學(xué)雜志;2006年02期

2 艾菁,王麗梅,夏威,耿美玉;Tat蛋白結(jié)構(gòu)與功能的研究進展[J];細胞與分子免疫學(xué)雜志;2005年S1期

3 王瑩瑩;龔莉;王成港;王春龍;;丙型肝炎治療新藥替拉瑞韋[J];藥物評價研究;2011年06期

4 徐芹;董金華;;Boceprevir[J];中國藥物化學(xué)雜志;2011年05期

5 張繼明,周紅霞;丙型肝炎病毒的復(fù)制周期及抗病毒藥物的靶位[J];肝臟;2004年S1期

本文編號：2397844