一种新型的真核表达载体的IFN -α介导的抗HBV作用

StephenW

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-alpha

Haotian Yu, Zhaohua Hou, Qiuju Han, Cai Zhang and Jian Zhang       

Haotian Yu1
Email: [email protected]
Zhaohua Hou1
Email: [email protected]
Qiuju Han1
Email: [email protected]
Cai Zhang1
Email: [email protected]
Jian Zhang1*
* Corresponding author
Email: [email protected]
1 Institute of Immunopharmacology and Immunotherapy, School of
Pharmaceutical Sciences, Shandong University, 44 Wenhua West Road, Jinan
250012, China

Virology Journal 2013, 10:270 doi:10.1186/1743-422X-10-270
Published: 29 August 2013
Abstract (provisional)
Background

Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-alpha) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-alpha at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-alpha) in vitro was carried out.
Methods

HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-alpha or empty plasmid by using LipofectamineTM 2000 reagent. The expression levels of IFN-alpha were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-alpha on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-alpha on IFN-alpha-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-alpha and the combination with lamivudine on HBV were also examined.
Results

pSecTagB-IFN-alpha could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-alpha-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-alpha, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-alpha vector revealed effectively anti-HBV effect than exogenously added IFN-alpha. Moreover, lamivudine combined with endogenously expressed IFN-alpha exhibited stronger anti-HBV effect than with exogenous IFN-alpha.
Conclusion

Our results showed that endogenously expressed IFN-alpha can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-alpha.

StephenW

一种新型的真核表达载体的IFN -α介导的抗HBV作用

昊天玉，昭化后，秋菊汉，张彩和张謇

昊天宇1
电子邮件： yu.ht8805 @ gmail.com
昭化Hou1的
电子邮件： [email protected]的
秋菊涵1
电子邮件： [email protected]的
蔡章1
电子邮件： [email protected]的
建张心*
*通讯作者
电子邮件： [email protected]的
免疫药理学，免疫学研究所
药学院，山东大学，济南市文化西路44
250012 ，中国

病毒学杂志2013 ， 10:270 DOI ： 10.1186/1743-422X-10-270
发布时间： 2013年8月29日
摘要（临时）
背景

慢性B型肝炎是肝脏相关死亡的主要原因。 α-干扰素（ IFN -α ）能够抑制嗜肝DNA病毒的复制，并在适当的电平的持续稳定的IFN -α的表达可能是有益的HBV清除。随着分子克隆技术的发展，基因治疗中起着越来越重要的作用，在临床实践中。根据研究结果，试图探讨抗HBV作用介导的真核表达质粒（ pSecTagB - IFN -α ）在体外进行。
方法

HBV阳性HepG2.2.15细胞及其亲本细胞HepG2与pSecTagB - IFN -α或使用LipofectamineTM 2000试剂空质粒转染。 IFN -α的表达水平，通过逆转录聚合酶链反应（RT-PCR ）和酶联免疫吸附方法确定。 HBV的mRNA ， DNA和抗原pSecTagB - IFN -α的影响进行了分析，通过实时荧光定量PCR （定量RT - PCR ）和酶联免疫吸附试验。 RT - PCR ，定量RT - PCR和Western blot调查的影响pSecTagB - IFN -α ， IFN -α诱导的信号通路。此外，通过实时定量RT -PCR和ELISA检测，内源性表达的α-干扰素联合拉米夫定对HBV的抑制作用还审议。
结果

pSecTagB - IFN -α在肝癌细胞中高效表达，抑制HBV复制的HBV S基因（氢键）和HBV C基因（ HBc阳性）的mRNA ， HBV DNA负荷减少的减少，其特征在于，低内容B型肝炎表面抗原（HBsAg）和乙肝e抗原（ HBeAg）消失。机理研究表明， Janus激酶（ JAK ）的活化信号转导和转录（ STAT ）信号通路活化，上调IFN -α诱导的抗病毒效应和双链（DS） RNA检测的受体通过提供pSecTagB IFN- α，可能是负责为这些现象。此外， pSecTagB - IFN -α向量显示有效的抗乙肝病毒效果比外加IFN -α 。此外，拉米夫定结合内源性表达的IFN -α的比带外源性IFN -α的表现出更强的抗HBV效果。
结论

我们的研究结果表明，内源性表达IFN -α能有效地持续抑制乙型肝炎病毒复制的乙肝病毒感染的细胞。这些观测结果开了一个有前途的方式来设计新的抗病毒基因工程干扰素- α药物的基础上。

咬牙硬挺

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