针对乙型肝炎病毒前S2基因核酶的设计及体外切割活性研究  

Synthesis and preliminary study on cleavage activity of ribozyme to hepatitis B virus preS2 gene in vitro

作　　者：徐东平[1] 韩凤连[1] 施红雷 周云 金磊[1] 刘妍[1] 

机构地区：[1]解放军302医院生物工程研究室

出　　处：《中华实验和临床病毒学杂志》1998年第2期107-110,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的体外研究核酶对乙型肝炎病毒（ＨＢＶ）前Ｓ２基因的作用。方法设计合成一针对ＨＢＶａｙｗＤＮＡ前Ｓ２区第１１０，１２２和１３２位碱基的三靶位串联锤头状核酶基因，体外转录核酶基因和靶基因并进行切割实验；将核酶基因与逆转录病毒重组，转导２．２．１５细胞，观察转导细胞聚人血清白蛋白受体（ｐＨＳＡ－Ｒ）的表达水平。结果核酶在体外可有效切割靶基因转录物，在转入２．２．１５细胞后４周内对ｐＨＳＡ－Ｒ表达抑制率为５１．４５±４．５７％～４１．０１±４．１６％。结论本文设计合成的针对乙型肝炎病毒前Ｓ２基因的核酶在体外具有相应的生物学活性。In this paper,a multi-target hammerhead ribozyme gene was synthesized directed against 110,122 and 132 sites of nucleotide of HBV preS2 gene.The target gene fragment was cut from HBV genome containing plasmid pCP10.Both of the ribozyme and the target gene fragments were cloned into pGEM3Zf(-)plasmid and sequenced by dideoxy chain termination method.The transcription of both gene fragments was performed in vitro utilizing T7 RNA promoter in pGEM3Zf(-)plasmid.The cleavage activity of ribozyme to substrate was confirmed in vitro. For further evaluating intracellular function of ribozyme,two ribozyme-retroviral recombinant plasmids with different promoter type pDOR-ripc and tRNA-ripc were constructed.Pseudo-virus was collected through routine packaging procedure and transduted into 2.2.15 cells.RIA data showed a stable inhibition of pHSA-R antigen expression to the lowest extent of 41.01%±4.16 and the highest extent of 51.45%±4.57 within 4 weeks after transduction.No influence,however,on HBsAg and HBeAg expression was demonstrated after ribozyme gene transfer.

关 键 词：乙型肝炎病毒 核酶 基因治疗 

分 类 号：R373.21[医药卫生—病原生物学]