一种能够支持B型肝炎病毒复制的构建体

[A construct competent to support the replication of hepatitis B virus of genotype B].

作者信息

Li Lei, Zhang Zhen-Hua, Shen Hong

机构信息

Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Zhonghua Gan Zang Bing Za Zhi. 2009 Jan;17(1):16-20.

PMID:19203445

Abstract

OBJECTIVE

To construct a vector that is competent to support the replication of hepatitis B virus (HBV) of genotype B.

METHODS

The HBV genome of genotype B was amplified by PCR and ligated into pBlueskript II KS(+) vector, the resulting plasmid was verified by enzyme digestion and DNA sequencing. After transfection of this plasmid into Huh7 cells, the HBsAg and HBeAg antigens in culture medium were quantified by ELISA, the transcripts and replication intermediates of HBV were detected by northern blot and southern blot respectively. On the other hand, the plasmid was hydrodynamically injected into BALB/cJ mice via tail vein. Then the HBV DNA in serum was quantified by real-time PCR, and HBcAg expression in liver tissue was detected by immunohistochemistry.

RESULTS

After transfection of the plasmid into Huh7 cells, the HBsAg and HBeAg antigens were detected in the culture medium, the transcripts and replication intermediates of HBV were detected in the cells. High titer of HBV DNA was detected in the serum of hydrodynamic-injected mice. Immunostaining indicated that HBcAg was expressed in hepatocytes of injected mice.

CONCLUSION

This construct is competent to support the replication of hepatitis B virus of genotype B.

摘要

目的

构建一种能够支持B型乙型肝炎病毒（HBV）复制的载体。

方法

通过PCR扩增B型HBV基因组，并将其连接到pBlueskript II KS(+)载体中，通过酶切和DNA测序验证所得质粒。将该质粒转染至Huh7细胞后，采用ELISA法对培养基中的HBsAg和HBeAg抗原进行定量，分别采用Northern印迹法和Southern印迹法检测HBV的转录本和复制中间体。另一方面，通过尾静脉将该质粒经流体动力学注射到BALB/cJ小鼠体内。然后采用实时PCR法对血清中的HBV DNA进行定量，采用免疫组织化学法检测肝组织中的HBcAg表达。