通过引入连接子增强对乙肝病毒靶向核糖核酸酶的抑制作用。

Inhibition of HBV targeted ribonuclease enhanced by introduction of linker.

作者信息

Gong Wei-Dong, Liu Jun, Ding Jin, Zhao Ya, Li Ying-Hui, Xue Cai-Fang

机构信息

Department of Pathogenic Organisms, Fourth Military Medical University, Xi'an 710033, Shaanxi Province, China.

出版信息

World J Gastroenterol. 2003 Jul;9(7):1504-7. doi: 10.3748/wjg.v9.i7.1504.

DOI:10.3748/wjg.v9.i7.1504

PMID:12854151

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4615492/

Abstract

AIM

To construct human eosinophil-derived neurotoxin(hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly(4)Ser) (3) between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro.

METHODS

Previously constructed pcDNA3.1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL. Meanwhile, metabolism of cells was evaluated by MTT colorimetry.

RESULTS

hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly(4)Ser)(3) between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3.1(-)/TR (P=0.036, P<0.05). MTT assay suggested that there were no significant differences between groups (P=0.08, P>0.05).

CONCLUSION

Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.

摘要

目的

构建人嗜酸性粒细胞衍生神经毒素（hEDN）与乙肝病毒核心蛋白（HBVc）的真核融合表达载体，并在两者之间引入连接肽（Gly(4)Ser）(3)以优化分子折叠，用于体外抑制乙肝病毒复制。

方法

以先前构建的pcDNA3.1(-)/TR为模板。合成连接肽序列并退火形成双链连接肽，克隆至pcDNA3.1(-)/TR中构建质粒pcDNA3.1(-)/HBc-连接肽。然后通过PCR扩增hEDN片段并插入pcDNA3.1(-)/HBc-连接肽中，构建成pcDNA3.1(-)/TNL，效应分子与靶分子通过连接肽序列分隔。采用间接免疫荧光染色鉴定pcDNA3.1(-)/TNL的表达。用放射免疫分析法分析pcDNA3.1(-)/TNL的抗乙肝病毒活性。同时，采用MTT比色法评估细胞代谢情况。

结果

成功构建了hEDN与HBVc之间含连接肽（Gly(4)Ser）(3)的真核融合表达载体。pcDNA3.1(-)/TNL在HepG2.2.15细胞中高效表达。与pcDNA3.1(-)/TR相比，pcDNA3.1(-)/TNL转染细胞的HBsAg浓度显著降低（P = 0.036，P<0.05）。MTT分析表明各组间无显著差异（P = 0.08，P>0.05）。