靶向核糖核酸酶抗乙肝病毒作用的研究和抗乙肝病毒机制的初步探索
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摘要
乙肝病毒感染是一个世界范围的卫生问题。据统计,全世界大约有3.5亿乙型肝炎病毒慢性感染者,我国约有1.2亿,其中相当大的一部分,特别是围产期/幼年期乙肝病毒感染者会迁延转变成为慢性肝炎,肝硬化或者肝癌。在过去的二十年中,虽然疫苗计划免疫降低了急、慢性乙肝病毒感染的发生率,但是现有的数目庞大的感染人群、约5%的对疫苗无应答者以及逃避疫苗诱导免疫的乙肝病毒突变株,都表明急需有效的治疗手段。对慢性乙型肝炎感染者的治疗,目前临床应用的α干扰素和核苷类似物有一定疗效,但是干扰素有效率不高(约为30%-40%),耐受性差,价格昂贵,副作用和禁忌症都较多;核苷类似物包括lamivudine、famciclovir等停药后部分患者的病情会复发,而长期用药又会导致耐药株的出现。因此,目前的治疗乙肝病毒感染的方法并不令人满意,迫切需要探索新的有效的治疗方法。近年来,随着生物技术的发展,基因治疗的研究取得了令人瞩目的成就,这也为乙型肝炎的治疗开辟了新的途径。
1991年,Natsoulis和Boeke在Nature杂志首次提出一种新的抗病毒的策略:核衣壳导向的病毒灭活(capsid-targeted viral inactivation,CTVI)并将其应用于逆转录病毒的实验治疗。乙肝病毒的复制过程类
Hepatitis B vims (HBV) infection is still a major health concern around the world. It has been established that more than 350 million people are infected by HBV and 120 million in our country, and a lot of them will evolve into chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, especially those who have infected in the childhood. Past 20 years, vaccine injected have decreased acute and chronic HBV infection, but there are so much people infected and 5% non-responders for vaccine and mutants for escaping immunization, which all need the effective antiviral treatment. To date, interferon- α (IFN- α ), an immuno- modulator, and two synthetic nucleoside analogues, lamivudine and adefovir dipivoxil, are the only licensed antiviral agents for the treatment of chronic HBV infection. However, IFN- α treatment response in chronic HBV infection patients rates range between 30% and 40%, and IFN-α therapy is very expensive and associated with a number of adverse effects, such as flu-like symptoms, paracetamol, fatigue, leucopoenia and depression, and so on. Nucleotide analogues such as lamivudine can inhibit replication of HBV via inhibiting the synthesis of minus-strand DNA of HBV, but cease of the treatment
usually leads to relapse of the infection and drug-resistant virus variants have emerged. In a word, the limited efficacy of current treatment for HBV infection justifies the search for new treatment strategy. The remarkableacheivements in the gene therapy bring us wishes to cure this disease.Capsid-targeted viral inactivation (CTVI), first proposed by Natsoulis and Boeke in 1991, is a conceptually powerful antiviral approach. The replication of Hepadnavirus, including HBV, is unique in DNA virus in that it needs a RNA intermediate and a reverse transcription process. This 3.5 kb RNA intermediate contains all genetic information of HBV and is so called pregenomic RNA (pgRNA). The pgRNA is first translated to both the capsid, or core, protein (c protein) and the DNA polymerase protein (P protein). Then the pgRNA is bound first by P protein and cellular chaperones to form a complex which is then encapsidated by C protein. Inside the nucleocapsids, P protein catalyzes the synthesis of minus-strand DNA via reverse transcription and then the incomplete plus-strand DNA. The replication characteristic of HBV hints CTVI can also be applied to this malicious virus.Previously, we reported the construction of a fusion protein of HBV C protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN) and other control plasmid, including including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-->Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, we found that the targeted ribonuclease could inhibit HBV replication in vitro. The aim of this research is to detect further antiviral effect by targeted ribonuclease and to explore its antiviral mechanism.First, Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was
performed. After that, RT-PCR was used to verify the transgene expression. Then, all the plasmid and pCMV.SPORT- & -gal were co-transfected into 2.2.15 cells. After 48 h, we prepared the cell extract of every co-transfection group to compare the efficiency each other. Under the condition of verifying the transgene expression and no difference in the transfection efficiency, we detected the further antiviral effect of targeted ribonuclease. Concentration of HBsAg and HBeAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay. The concentration of HBsAg and HBeAg in the supernatant of 2.2.15 cells transfected with p/TR was significantly lower than controls, Compared with that of mock transfected 2.2.15 cells, the concentration of HBsAg in the supernatant of 2.2.15 cells transfected with p/TR was decreased by 51% and 61%, respectively. Concentration of HBsAg and HBeAg of the transfected cells was measured using HBsAg and HBeAg diagnosis kit. The concentration of HBsAg and HBeAg of 2.2.15 cells transfected with p/TR was significantly lower than controls, Compared with that of mock transfected 2.2.15 cells, the concentration of HBsAg in the supernatant of 2.2.15 cells transfected with p/TR was decreased by 31% and 41%, respectively. HBV DNA copies were determined by fluorescent real time PCR. The significant decrease of HBV DNA copies in p/TR transfection, compared with that of mock transfected 2.2.15 cells, showed the anti-HBV activity of the targeted ribonuclease.Here before, we verify the anti-HBV activity of the targeted ribonuclease. To explore the anti-HBV mechanism, we expressed the active targeted ribonuclease and control protein in E.coli, detecting their ribonuclease activity and studying their targeted degrading HBV pgRNA in and out 2.2.15 cells. First, the coding genes TR, TRmut and hEDN in the p/TR, p/TRmut and p/hEDN were cloned into prokaryotic expression vector pET32a(+). After digesting with BamHl and Hindlll, we could verify the
successful construct of pET32a(+)/TR, pET32a(+)/TRmut and pET32a(+)/hEDN. All the recombinant prokaryotic expression vectors were transformed into E.coli BL21, inducing by different IPTG concentration and different induction time. The expression products were determined by SDS-PAGE and Western blot was used to confirm the target protein expression. Results showed that, under different induction conditions, the the recombinant prokaryotic expression vectors pET32a(+)/TR, pET32a(+)/TRmut and pET32a(+)/hEDN were successfully induced to express in E.coli. These three kinds of expression products with the molecular weight were 43kD, 43kD and 25kD, respectively. Western blot indicated a single band in the corresponding location. The expression products mainly existed in a form of inclusion bodies. pET32a(+)/TR was purified by Ni-NTA agarose under denaturing conditions, though some nonspecific binding proteins couldn't be excluded from the purified product. After the purification, TR was refolded by large volume dialysis and concentrated by by Ni-NTA agarose under native conditions. For pET32a(+)/TRmut and pET32a(+)/hEDN, we used the conventional inclusion body method to purify and slowly dialysis to refold. The refolding protein was freeze-dried to store. Using the yeast tRNA as a substrate, the analysis of ribonuclease activity showed that the results of refolding is very good.In other to detect the ribonuclease activity of the refolding protein, the qualitative ribonuclease activity analysis of refolding protein was done. The liver RNA of mouse was extracted by Trizol reagent and used as the substrate. After the incubation of the liver RNA and TR, the degrading of RNA was analyzed by RNA electrophoresis. The results showed that the two binds of RNA incubating with TRmut were very clear and that of incubating with TR were degraded to not so clear and showed the dose-dependent. Then, the quantitative ribonuclease activity analysis of refolding protein was
1991年,Natsoulis和Boeke在Nature杂志首次提出一种新的抗病毒的策略:核衣壳导向的病毒灭活(capsid-targeted viral inactivation,CTVI)并将其应用于逆转录病毒的实验治疗。乙肝病毒的复制过程类
Hepatitis B vims (HBV) infection is still a major health concern around the world. It has been established that more than 350 million people are infected by HBV and 120 million in our country, and a lot of them will evolve into chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, especially those who have infected in the childhood. Past 20 years, vaccine injected have decreased acute and chronic HBV infection, but there are so much people infected and 5% non-responders for vaccine and mutants for escaping immunization, which all need the effective antiviral treatment. To date, interferon- α (IFN- α ), an immuno- modulator, and two synthetic nucleoside analogues, lamivudine and adefovir dipivoxil, are the only licensed antiviral agents for the treatment of chronic HBV infection. However, IFN- α treatment response in chronic HBV infection patients rates range between 30% and 40%, and IFN-α therapy is very expensive and associated with a number of adverse effects, such as flu-like symptoms, paracetamol, fatigue, leucopoenia and depression, and so on. Nucleotide analogues such as lamivudine can inhibit replication of HBV via inhibiting the synthesis of minus-strand DNA of HBV, but cease of the treatment
usually leads to relapse of the infection and drug-resistant virus variants have emerged. In a word, the limited efficacy of current treatment for HBV infection justifies the search for new treatment strategy. The remarkableacheivements in the gene therapy bring us wishes to cure this disease.Capsid-targeted viral inactivation (CTVI), first proposed by Natsoulis and Boeke in 1991, is a conceptually powerful antiviral approach. The replication of Hepadnavirus, including HBV, is unique in DNA virus in that it needs a RNA intermediate and a reverse transcription process. This 3.5 kb RNA intermediate contains all genetic information of HBV and is so called pregenomic RNA (pgRNA). The pgRNA is first translated to both the capsid, or core, protein (c protein) and the DNA polymerase protein (P protein). Then the pgRNA is bound first by P protein and cellular chaperones to form a complex which is then encapsidated by C protein. Inside the nucleocapsids, P protein catalyzes the synthesis of minus-strand DNA via reverse transcription and then the incomplete plus-strand DNA. The replication characteristic of HBV hints CTVI can also be applied to this malicious virus.Previously, we reported the construction of a fusion protein of HBV C protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN) and other control plasmid, including including p/hEDN, p/HBVc, and p/TNmut in which a Lys113-->Arg mutation was introduced by sequential PCR to eliminate the ribonuclease activity of hEDN, were also constructed. Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was performed. After that, we found that the targeted ribonuclease could inhibit HBV replication in vitro. The aim of this research is to detect further antiviral effect by targeted ribonuclease and to explore its antiviral mechanism.First, Liposome-mediated transfection of 2.2.15 cells by p/TN, p/TNmut, p/hEDN, p/HBVc, and pcDNA3.1 (-), or mock transfection was
performed. After that, RT-PCR was used to verify the transgene expression. Then, all the plasmid and pCMV.SPORT- & -gal were co-transfected into 2.2.15 cells. After 48 h, we prepared the cell extract of every co-transfection group to compare the efficiency each other. Under the condition of verifying the transgene expression and no difference in the transfection efficiency, we detected the further antiviral effect of targeted ribonuclease. Concentration of HBsAg and HBeAg in the supernatant of the transfected cells was measured using solid-phase radioimmunoassay. The concentration of HBsAg and HBeAg in the supernatant of 2.2.15 cells transfected with p/TR was significantly lower than controls, Compared with that of mock transfected 2.2.15 cells, the concentration of HBsAg in the supernatant of 2.2.15 cells transfected with p/TR was decreased by 51% and 61%, respectively. Concentration of HBsAg and HBeAg of the transfected cells was measured using HBsAg and HBeAg diagnosis kit. The concentration of HBsAg and HBeAg of 2.2.15 cells transfected with p/TR was significantly lower than controls, Compared with that of mock transfected 2.2.15 cells, the concentration of HBsAg in the supernatant of 2.2.15 cells transfected with p/TR was decreased by 31% and 41%, respectively. HBV DNA copies were determined by fluorescent real time PCR. The significant decrease of HBV DNA copies in p/TR transfection, compared with that of mock transfected 2.2.15 cells, showed the anti-HBV activity of the targeted ribonuclease.Here before, we verify the anti-HBV activity of the targeted ribonuclease. To explore the anti-HBV mechanism, we expressed the active targeted ribonuclease and control protein in E.coli, detecting their ribonuclease activity and studying their targeted degrading HBV pgRNA in and out 2.2.15 cells. First, the coding genes TR, TRmut and hEDN in the p/TR, p/TRmut and p/hEDN were cloned into prokaryotic expression vector pET32a(+). After digesting with BamHl and Hindlll, we could verify the
successful construct of pET32a(+)/TR, pET32a(+)/TRmut and pET32a(+)/hEDN. All the recombinant prokaryotic expression vectors were transformed into E.coli BL21, inducing by different IPTG concentration and different induction time. The expression products were determined by SDS-PAGE and Western blot was used to confirm the target protein expression. Results showed that, under different induction conditions, the the recombinant prokaryotic expression vectors pET32a(+)/TR, pET32a(+)/TRmut and pET32a(+)/hEDN were successfully induced to express in E.coli. These three kinds of expression products with the molecular weight were 43kD, 43kD and 25kD, respectively. Western blot indicated a single band in the corresponding location. The expression products mainly existed in a form of inclusion bodies. pET32a(+)/TR was purified by Ni-NTA agarose under denaturing conditions, though some nonspecific binding proteins couldn't be excluded from the purified product. After the purification, TR was refolded by large volume dialysis and concentrated by by Ni-NTA agarose under native conditions. For pET32a(+)/TRmut and pET32a(+)/hEDN, we used the conventional inclusion body method to purify and slowly dialysis to refold. The refolding protein was freeze-dried to store. Using the yeast tRNA as a substrate, the analysis of ribonuclease activity showed that the results of refolding is very good.In other to detect the ribonuclease activity of the refolding protein, the qualitative ribonuclease activity analysis of refolding protein was done. The liver RNA of mouse was extracted by Trizol reagent and used as the substrate. After the incubation of the liver RNA and TR, the degrading of RNA was analyzed by RNA electrophoresis. The results showed that the two binds of RNA incubating with TRmut were very clear and that of incubating with TR were degraded to not so clear and showed the dose-dependent. Then, the quantitative ribonuclease activity analysis of refolding protein was
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