DHBV侵染规律和preS蛋白原核表达及在评价抗人类乙肝新药的应用
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摘要
鸭乙型肝炎病毒(Duck Hepatitis B Virus,DHBV)与乙型肝炎病毒(Hepatitis BVirus,HBV)同属嗜肝DNA病毒科,这类病毒的特征是有强的嗜肝细胞特性,在病毒形态、基因组结构、复制过程等生物学特性相似。DHBV感染鸭模型是研究HBV的生物学特性、致病机制和抗HBV药物的实验动物模型。本文通过调查四川地区麻鸭自然携带DHBV的情况,分离DHBV毒株,以此毒株为模板,扩增主要抗原基因preS。通过构建DHBV-preS原核表达质粒并诱导表达,从而对表达蛋白免疫原性、表达蛋白抗体介导的免疫组化检测人工感染DHBV后在体内的蛋白定位分析与侵染规律,以期系统地了解的入侵方式和复制机理,为阐明DHBV的发病机制提供关键的实验数据。同时建立基于定量检测DHBV的FQ-PCR方法,用于DHBV疾病模型研究,并结合体外模型HepG2.2.15细胞研究抗乙型肝炎新药,结果如下:
DHBV毒株的分离及分子特征解析结果表明:四川麻鸭自然携带DHBV 4%,证实四川麻鸭是研究实验性DHBV感染动物模型的良好鸭种;对分离到的其中3株DHBV阳性血清全基因克隆、测序并进行生物信息学分析,发现全基因组均含3006个核苷酸(GenBank登录号EU429324,EU429325,EU429326),具有X-like开放阅读框特征;进一步研究ORF-S还表明该基因编码33个氨基酸,有四个疏水区,无N-糖基化位点,也无豆蔻酰化位点,在1～30aa内有一个明显的信号肽,在19～20aa处有断点,跨膜螺旋预测为外膜蛋白,抗原位点主要分布于preS区氨基酸序列中。
根据DHBV-preS序列,设计一对特异性引物,以分离的DHBV毒株为模板扩增目标基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将DHBV-preS基因正向插入原核表达载体pET-32a(+)的ApaI和NcoI位点间,成功构建了重组表达质粒pET32a(+)/DHBV-preS。重组表达质粒pET32a(+)/DHBV-preS转化表达宿主菌BL21(DE3),用IPTG诱导,能表达出了大小约为37kD的preS重组蛋白,与预期表达蛋白分子量大小相符;经对不同诱导时间及诱导剂IPTG浓度等条件进行优化,确定重组质粒pET32a(+)/DHBV-preS的最佳诱导条件为0.8mmol/LIPTG、37℃条件下诱导4h。表达产物用镍柱亲和层析纯化后,将得到的preS重组蛋白做梯度稀释,初步建立ELISA检测DHBsAb的方法(间接法);同时将纯化的重组蛋白与等量弗氏佐剂混合制备preS重组蛋白免疫原,四次免疫家兔,获得的兔抗DHBV-preS高免血清经辛酸-硫酸铵粗提后,阴离子交换柱层析纯化抗体IgG。
建立DHBV-preS基因表达蛋白抗体介导的免疫组化方法;针对DHBV的保守序列设计并合成引物及荧光标记探针,建立实时荧光定量聚合酶链反应(fluorsescencequantitative polymerase chain reaction,FQ-PCR)检测方法。建立的标准曲线循环阈值(cycle threshold,Ct值)与模板浓度具有良好的线性关系,相关系数为0.993,将其检测极限的Ct值通过标准曲线换算成拷贝数约为5copies/L,未检测到鸭病毒性肝炎、鸭瘟病毒、鸭源致病性大肠埃希氏菌、鸭源致病性沙门氏菌、鸭疫里默氏杆菌,表明FQ-PCR检测DHBV方法灵敏、特异性强。
采用鸭乙型肝炎病毒人工方法感染1日龄四川麻鸭,攻毒后于不同时间采血分离血清,采集肝脏、胰腺、肾脏、脾脏等组织或器官,经建立的DHBV-preS基因表达蛋白抗体免疫组化方法和FQ-PCR方法检测DHBV在感染鸭体内侵染过程与定位分布,并结合组织学和血液生化指标对DHBV在感染鸭体模型侵染规律进行系统观察。结果显示:感染后2d可在血清和肝组织中检测出DHBV DNA,血清中DHBV DNA从感染后第10d～30d DHBV DNA的拷贝数保持在1.00E+09以上,肝组织中DHBVDNA的含量从感染后第5d～52d保持在5.00E+09以上;肝脏DHBV DNA第14d达到最高水平,而血清中DHBV DNA第22d达到最高水平。肝脏、胰腺、肾脏、脾脏的损伤程度与DHBV DNA水平成正相关;在感染后3～52d内的不同时间点从肝脏、胰腺、肾脏、脾脏及大脑六种组织器官中检测出DHBsAg,DHBsAg主要分布在细胞浆,少部分分布于细胞核,未能从食道、腺胃、肺、心脏、生殖器和肌肉中检测到DHBsAg,表明DHBV嗜肝的同时具有泛嗜性,且在靶器官的侵染与分布具有一定选择性;在感染后1～3d、52d以后各组织相继不能检出DHBsAg,感染后12～31d病毒在机体内各组织的分布最为广泛,感染后4d肝脏开始出现DHBsAg阳性细胞,感染后6d肾脏、胰腺、脾脏相继开始出现DHBsAg阳性细胞,而脑组织在感染后10d出现阳性细胞;肝脏的检出率最高,持续至52d,大脑的检出率最低,分析表明该蛋白可能是膜蛋白,具有运输核浆与引导病毒组分进入核内参与病毒复制的功能。DHBV感染后第4d,总蛋白和白蛋白含量开始降低,谷丙、谷草转氨酶活性升高(P＜0.01),乳酸脱氢酶活性升高(P＜0.01),肌酐和尿素有变化但无规律,表现为升高趋势,感染后44d开始,各项血液生化指标逐渐趋于正常值,表明DHBV引起肝脏损伤的同时累及肾脏。
在建立的抗人类乙型肝炎新药体内药效评价的技术平台和体外模型HepG2.2.15上评价抗乙型肝炎新研制药物-核苷类似物(代号PNA)的作用。在HepG2.2.15细胞系中,PNA有明确的抑制HBV DNA复制作用,抑制HBsAg和HBeAg,在7.8～62.5μg/mL剂量范围内有剂量—时间依赖关系,未见到明显细胞学毒性;在鸭乙肝动物模型中,PNA对DHBV DNA抑制率达50%的最低用药剂量为口服20mg/kg,口服40mg/kg、80mg/kg PNA对DHBV的抑制率与拉米夫定口服组(50mg/kg)基本一致,可以减轻鸭脏炎症,抑制DHBsAg在肝脏中的表达。
Hepatitis B virus(HBV)is a human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma.The long-term objectives of our research are to design therapeutic strategies and to improve vaccines for human HBV infection.Our understanding of the immune response to HBV is incomplete,largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models.Much of our current understanding of the viral life cycle of HBV infection is derived from studies of duck HBV(DHBV)infection in their natural hosts.Although the duck model has recognized limitations,it has proven to be an exceptionally useful system for the elucidation of hepadnaviral replication strategy and antiviral drug screening.
One of the major advantages of the DHBV model is that its natural host,domestic duck,is inexpensive,easy to handle,and available from commercial breeders.It is necessary to develop a standard DHBV animal model to enhance our understanding of the replication strategy,natural history,and pathogenesis of HBV infection.So the invasion procedure of DHBV infection in model should be clarified.The contents was summaried as follows:
1.Molecular characterization of DHBV isolates from Sichuan Mallard.To clone and analyze the genome of a duck hepatitis B virus(DHBV)isolated from a Sichuan Mallard,DHBV DNA-positive serum were collected from the investigation of the natural carrying rate of DHBV in adult ducks in Sichuan area.The complete genome of a DHBV strain was amplified by polymerase chain reaction(PCR)and sequencing.The natural carrying rate of DHBV in Sichuan adult ducks was 4%.The strain had DHBV genomes of 3,006 nucleotides with unique characteristic features.The present investigation shows Sichuan ducks are one of the best species for experimental DHBV infection.
2.Expression preS protein in Ecoli.and preparation anti-preS antibody.To purify the recombinant preS peptide of duck hepatitis B surface antigen in E.coli and to investigate its physiochemical characters and immunogenicity.The PreS gene was amplified by PCR,and then inserted into prokaryotic expression vector pET32a(+).The recombinant plasmid was confirmed by sequence analysis and named pET32a(+) /DHBV-preS.The expression of recombinant proteins were induced with IPTG,and then the expression product were confirmed by Western blot analysis and further purified by affinity chromatography methods.Recombinant proteins of PreS displayed bands of Mr 37,000 on SDS-PAGE gel.The purity of the fusion protein was over 90%after purification by affinity chromatography method.The specific antibody titer could reach 1:32 in recombinant protein immunized rabbit.Purified fusion proteins laid a foundation for better understanding of the mechanism of DHBV PreS protein in viral endocytosis and were helpful for seeking the PreS-related protein.
3.Quantification of DHBV by fluorescence quantitative PCR(FQ-PCR).To develop a fluorescence quantitative PCR based on TaqMan chemistry for quantification of DHBV.The PCR fragment of DHBV DNA was cloned into vector pMD18-T.The recombinant plasmid was purified and subsequently quantified as DHBV DNA strand.A pair of primers and fluorescent probe were designed from conserved sequence.The experimental conditions and reagents of amplification were sopisticatedly optimized in order to produce perfect amplification efficiency and reduce non-specific amplification. The standard curve indicated the linear relationship between CT(cycle threshold)and template concentration with a good correlation(r=0.993),sensitivity was 5 copies/L of DHBV genome.The FQ-PCR method for quantification of DHBV DNA is a simple,highly sensitive and specific method.
4.Immunohistochemical assay for detection DHBsAg.To establish immune-ohistochemical assay for duck hepatitis surface antigen(DHBsAg)using anti-preS antibody.Liver,Pancreas,Kidney,Spleen,Cerebrum samples,taken from 12 days ducklings post-inoculation,were used to locate DHBsAg by IHC.The result illustrated that DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining;Positive cells also were detected in Liver,Pancreas,Kidney,Spleen and Cerebrum.The methodology is helpful to illustrate the localization of virus specific antigen in situ at subcellsular level and for exploring important aspects in this family of hepadnavirus such as viral invasion procedure,viral replication,course of infection, pathogenesis and the response to antiviral therapy.These data provide further information about the pathogenesis and distribution of DHBV infection for animal model.
5.The invasion procedure in DHBV infection model.To develop a standard DHBV animal model and use it as an in vivo experimental system to study DHBV invasion prcedure and antiviral strategies.One-day-old ducklings were subcutaneously inoculated DHBV serum and sacrificed during in different time from 2d to 70d respectively whose tissues were collected including liver,pancreas,kidney,spleen,cerebrum et al.These tissues were detected using developed immunohistochemistry.Quantitative analysis of DHBV DNA in serum and liver samples was performed with FQ-PCR.Pathological changes were observed and biochemical indexes were determined at different times.The results showed that positive signals can be detected from liver,pancreas,kidney,spleen and cerebrum during different time from 3dpi to 52dpi.DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining.The most positive tissue samples was at 12d pi to 31dpi(post inoculation).DHBsAg positive signals appeared in liver at 4dpi,in kidney,pancreas and spleen at 6dpi.The max detection rate was the liver among the detected tissues while the min detection rate was the cerebrum.DHBsAg can not be detected from gullet,glandular stomach,lung,heart,genitals and muscle.Increase the serum levels of biochemical index,especially ALT,AST,SB,UN and CR.The liver and renal functions were changed in accordance with DHBV DNA level,pathological changes and DHBsAg changes.The Sichuan duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research.
6.Antiviral treatment to the HepG2.2.15 and DHBV-infected ducklings. Nucleoside analogues provide a large reservoir of potentially active anti-hepatitis B virus (HBV)agents.To evaluated the novel nucleoside analogue(PNA)in HepG2 2.2.15 cell line and in the duck model of hepatitis B.The extracellular HBV DNA,hepatitis B e antigen(HBeAg)and hepatitis B surface antigen(HBsAg)concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA,respectively.PNA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner.Consistent with the HBV antigen reduction,PNA also reduced the extracellular HBV DNA level in a dose-dependent manner between 7.8 and 62.5μg/mL.PNA(25,50,or 100mg/kg, intraperitoneally,twice daily)can significantly lower the DHBV DNA levels in sera and livers.Histopathological changes in the treatment groups were significantly improved and the changes were associated with liver viral DNA levels.Immunohistological staining of the liver confirmed the duck hepatitis B surface antigen(DHBsAg)reduction by PNA The relapse in PNA-treatment groups is slighter than in 3TC-treatment group at 3 day withdrawl,In conclusion,the results demonstrate that PNA is a strong inhibitor of anti-HBV activity both in vitro and in vivo.
DHBV毒株的分离及分子特征解析结果表明:四川麻鸭自然携带DHBV 4%,证实四川麻鸭是研究实验性DHBV感染动物模型的良好鸭种;对分离到的其中3株DHBV阳性血清全基因克隆、测序并进行生物信息学分析,发现全基因组均含3006个核苷酸(GenBank登录号EU429324,EU429325,EU429326),具有X-like开放阅读框特征;进一步研究ORF-S还表明该基因编码33个氨基酸,有四个疏水区,无N-糖基化位点,也无豆蔻酰化位点,在1～30aa内有一个明显的信号肽,在19～20aa处有断点,跨膜螺旋预测为外膜蛋白,抗原位点主要分布于preS区氨基酸序列中。
根据DHBV-preS序列,设计一对特异性引物,以分离的DHBV毒株为模板扩增目标基因并将其克隆至pMD18-T载体,经PCR、酶切和DNA测序鉴定后,将DHBV-preS基因正向插入原核表达载体pET-32a(+)的ApaI和NcoI位点间,成功构建了重组表达质粒pET32a(+)/DHBV-preS。重组表达质粒pET32a(+)/DHBV-preS转化表达宿主菌BL21(DE3),用IPTG诱导,能表达出了大小约为37kD的preS重组蛋白,与预期表达蛋白分子量大小相符;经对不同诱导时间及诱导剂IPTG浓度等条件进行优化,确定重组质粒pET32a(+)/DHBV-preS的最佳诱导条件为0.8mmol/LIPTG、37℃条件下诱导4h。表达产物用镍柱亲和层析纯化后,将得到的preS重组蛋白做梯度稀释,初步建立ELISA检测DHBsAb的方法(间接法);同时将纯化的重组蛋白与等量弗氏佐剂混合制备preS重组蛋白免疫原,四次免疫家兔,获得的兔抗DHBV-preS高免血清经辛酸-硫酸铵粗提后,阴离子交换柱层析纯化抗体IgG。
建立DHBV-preS基因表达蛋白抗体介导的免疫组化方法;针对DHBV的保守序列设计并合成引物及荧光标记探针,建立实时荧光定量聚合酶链反应(fluorsescencequantitative polymerase chain reaction,FQ-PCR)检测方法。建立的标准曲线循环阈值(cycle threshold,Ct值)与模板浓度具有良好的线性关系,相关系数为0.993,将其检测极限的Ct值通过标准曲线换算成拷贝数约为5copies/L,未检测到鸭病毒性肝炎、鸭瘟病毒、鸭源致病性大肠埃希氏菌、鸭源致病性沙门氏菌、鸭疫里默氏杆菌,表明FQ-PCR检测DHBV方法灵敏、特异性强。
采用鸭乙型肝炎病毒人工方法感染1日龄四川麻鸭,攻毒后于不同时间采血分离血清,采集肝脏、胰腺、肾脏、脾脏等组织或器官,经建立的DHBV-preS基因表达蛋白抗体免疫组化方法和FQ-PCR方法检测DHBV在感染鸭体内侵染过程与定位分布,并结合组织学和血液生化指标对DHBV在感染鸭体模型侵染规律进行系统观察。结果显示:感染后2d可在血清和肝组织中检测出DHBV DNA,血清中DHBV DNA从感染后第10d～30d DHBV DNA的拷贝数保持在1.00E+09以上,肝组织中DHBVDNA的含量从感染后第5d～52d保持在5.00E+09以上;肝脏DHBV DNA第14d达到最高水平,而血清中DHBV DNA第22d达到最高水平。肝脏、胰腺、肾脏、脾脏的损伤程度与DHBV DNA水平成正相关;在感染后3～52d内的不同时间点从肝脏、胰腺、肾脏、脾脏及大脑六种组织器官中检测出DHBsAg,DHBsAg主要分布在细胞浆,少部分分布于细胞核,未能从食道、腺胃、肺、心脏、生殖器和肌肉中检测到DHBsAg,表明DHBV嗜肝的同时具有泛嗜性,且在靶器官的侵染与分布具有一定选择性;在感染后1～3d、52d以后各组织相继不能检出DHBsAg,感染后12～31d病毒在机体内各组织的分布最为广泛,感染后4d肝脏开始出现DHBsAg阳性细胞,感染后6d肾脏、胰腺、脾脏相继开始出现DHBsAg阳性细胞,而脑组织在感染后10d出现阳性细胞;肝脏的检出率最高,持续至52d,大脑的检出率最低,分析表明该蛋白可能是膜蛋白,具有运输核浆与引导病毒组分进入核内参与病毒复制的功能。DHBV感染后第4d,总蛋白和白蛋白含量开始降低,谷丙、谷草转氨酶活性升高(P＜0.01),乳酸脱氢酶活性升高(P＜0.01),肌酐和尿素有变化但无规律,表现为升高趋势,感染后44d开始,各项血液生化指标逐渐趋于正常值,表明DHBV引起肝脏损伤的同时累及肾脏。
在建立的抗人类乙型肝炎新药体内药效评价的技术平台和体外模型HepG2.2.15上评价抗乙型肝炎新研制药物-核苷类似物(代号PNA)的作用。在HepG2.2.15细胞系中,PNA有明确的抑制HBV DNA复制作用,抑制HBsAg和HBeAg,在7.8～62.5μg/mL剂量范围内有剂量—时间依赖关系,未见到明显细胞学毒性;在鸭乙肝动物模型中,PNA对DHBV DNA抑制率达50%的最低用药剂量为口服20mg/kg,口服40mg/kg、80mg/kg PNA对DHBV的抑制率与拉米夫定口服组(50mg/kg)基本一致,可以减轻鸭脏炎症,抑制DHBsAg在肝脏中的表达。
Hepatitis B virus(HBV)is a human hepadnavirus that causes acute and chronic hepatitis and hepatocellular carcinoma.The long-term objectives of our research are to design therapeutic strategies and to improve vaccines for human HBV infection.Our understanding of the immune response to HBV is incomplete,largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models.Much of our current understanding of the viral life cycle of HBV infection is derived from studies of duck HBV(DHBV)infection in their natural hosts.Although the duck model has recognized limitations,it has proven to be an exceptionally useful system for the elucidation of hepadnaviral replication strategy and antiviral drug screening.
One of the major advantages of the DHBV model is that its natural host,domestic duck,is inexpensive,easy to handle,and available from commercial breeders.It is necessary to develop a standard DHBV animal model to enhance our understanding of the replication strategy,natural history,and pathogenesis of HBV infection.So the invasion procedure of DHBV infection in model should be clarified.The contents was summaried as follows:
1.Molecular characterization of DHBV isolates from Sichuan Mallard.To clone and analyze the genome of a duck hepatitis B virus(DHBV)isolated from a Sichuan Mallard,DHBV DNA-positive serum were collected from the investigation of the natural carrying rate of DHBV in adult ducks in Sichuan area.The complete genome of a DHBV strain was amplified by polymerase chain reaction(PCR)and sequencing.The natural carrying rate of DHBV in Sichuan adult ducks was 4%.The strain had DHBV genomes of 3,006 nucleotides with unique characteristic features.The present investigation shows Sichuan ducks are one of the best species for experimental DHBV infection.
2.Expression preS protein in Ecoli.and preparation anti-preS antibody.To purify the recombinant preS peptide of duck hepatitis B surface antigen in E.coli and to investigate its physiochemical characters and immunogenicity.The PreS gene was amplified by PCR,and then inserted into prokaryotic expression vector pET32a(+).The recombinant plasmid was confirmed by sequence analysis and named pET32a(+) /DHBV-preS.The expression of recombinant proteins were induced with IPTG,and then the expression product were confirmed by Western blot analysis and further purified by affinity chromatography methods.Recombinant proteins of PreS displayed bands of Mr 37,000 on SDS-PAGE gel.The purity of the fusion protein was over 90%after purification by affinity chromatography method.The specific antibody titer could reach 1:32 in recombinant protein immunized rabbit.Purified fusion proteins laid a foundation for better understanding of the mechanism of DHBV PreS protein in viral endocytosis and were helpful for seeking the PreS-related protein.
3.Quantification of DHBV by fluorescence quantitative PCR(FQ-PCR).To develop a fluorescence quantitative PCR based on TaqMan chemistry for quantification of DHBV.The PCR fragment of DHBV DNA was cloned into vector pMD18-T.The recombinant plasmid was purified and subsequently quantified as DHBV DNA strand.A pair of primers and fluorescent probe were designed from conserved sequence.The experimental conditions and reagents of amplification were sopisticatedly optimized in order to produce perfect amplification efficiency and reduce non-specific amplification. The standard curve indicated the linear relationship between CT(cycle threshold)and template concentration with a good correlation(r=0.993),sensitivity was 5 copies/L of DHBV genome.The FQ-PCR method for quantification of DHBV DNA is a simple,highly sensitive and specific method.
4.Immunohistochemical assay for detection DHBsAg.To establish immune-ohistochemical assay for duck hepatitis surface antigen(DHBsAg)using anti-preS antibody.Liver,Pancreas,Kidney,Spleen,Cerebrum samples,taken from 12 days ducklings post-inoculation,were used to locate DHBsAg by IHC.The result illustrated that DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining;Positive cells also were detected in Liver,Pancreas,Kidney,Spleen and Cerebrum.The methodology is helpful to illustrate the localization of virus specific antigen in situ at subcellsular level and for exploring important aspects in this family of hepadnavirus such as viral invasion procedure,viral replication,course of infection, pathogenesis and the response to antiviral therapy.These data provide further information about the pathogenesis and distribution of DHBV infection for animal model.
5.The invasion procedure in DHBV infection model.To develop a standard DHBV animal model and use it as an in vivo experimental system to study DHBV invasion prcedure and antiviral strategies.One-day-old ducklings were subcutaneously inoculated DHBV serum and sacrificed during in different time from 2d to 70d respectively whose tissues were collected including liver,pancreas,kidney,spleen,cerebrum et al.These tissues were detected using developed immunohistochemistry.Quantitative analysis of DHBV DNA in serum and liver samples was performed with FQ-PCR.Pathological changes were observed and biochemical indexes were determined at different times.The results showed that positive signals can be detected from liver,pancreas,kidney,spleen and cerebrum during different time from 3dpi to 52dpi.DHBsAg was usually restricted to randomly scatter cells showing strong cytoplasmic staining.The most positive tissue samples was at 12d pi to 31dpi(post inoculation).DHBsAg positive signals appeared in liver at 4dpi,in kidney,pancreas and spleen at 6dpi.The max detection rate was the liver among the detected tissues while the min detection rate was the cerebrum.DHBsAg can not be detected from gullet,glandular stomach,lung,heart,genitals and muscle.Increase the serum levels of biochemical index,especially ALT,AST,SB,UN and CR.The liver and renal functions were changed in accordance with DHBV DNA level,pathological changes and DHBsAg changes.The Sichuan duck model with experimental DHBV infection of transfected supernatant is more suitable for the hepadnavirus biologic research.
6.Antiviral treatment to the HepG2.2.15 and DHBV-infected ducklings. Nucleoside analogues provide a large reservoir of potentially active anti-hepatitis B virus (HBV)agents.To evaluated the novel nucleoside analogue(PNA)in HepG2 2.2.15 cell line and in the duck model of hepatitis B.The extracellular HBV DNA,hepatitis B e antigen(HBeAg)and hepatitis B surface antigen(HBsAg)concentrations in cell culture medium were determined by quantitative real-time PCR and ELISA,respectively.PNA appeared to downregulate the secretion of HBsAg and HBeAg as well as the release of HBV DNA from HepG2 2.2.15 in a dose- and time-dependent manner.Consistent with the HBV antigen reduction,PNA also reduced the extracellular HBV DNA level in a dose-dependent manner between 7.8 and 62.5μg/mL.PNA(25,50,or 100mg/kg, intraperitoneally,twice daily)can significantly lower the DHBV DNA levels in sera and livers.Histopathological changes in the treatment groups were significantly improved and the changes were associated with liver viral DNA levels.Immunohistological staining of the liver confirmed the duck hepatitis B surface antigen(DHBsAg)reduction by PNA The relapse in PNA-treatment groups is slighter than in 3TC-treatment group at 3 day withdrawl,In conclusion,the results demonstrate that PNA is a strong inhibitor of anti-HBV activity both in vitro and in vivo.
引文
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