CAJ | 학술논문

目的构建对HCV基因组具有特异切割作用的新型靶向性核酶-M1GS。方法针对HCV基因组的保守区(5′UTR)设计并合成一段引导序列，通过PCR扩增直接将该引导序列连接于大肠杆菌核糖核酸酶P的催化亚基(M1 RNA)的3′末端，从而构建一种靶向性M1GS核酶。结果胞内切割实验表明，所构建的核酶(M1GS-HCV/C76)对HCV 5′UTR具有明显的切割作用，切割的位点在靶序列77~89 n t之间，属于特异性切割，具有胞内抗病毒活性。结论构建了一种对HCV 5′UTR具有靶向切割活性的M1GS核酶，且具有胞内抗病毒活性，为动物模型内抗病毒效应的评价提供了实验材料，为新型抗HCV药物的研究奠定了基础。
목적구건대HCV기인조구유특이절할작용적신형파향성핵매-M1GS。방법침대HCV기인조적보수구(5′UTR)설계병합성일단인도서렬，통과PCR확증직접장해인도서렬련접우대장간균핵당핵산매P적최화아기(M1 RNA)적3′말단，종이구건일충파향성M1GS핵매。결과포내절할실험표명，소구건적핵매(M1GS-HCV/C76)대HCV 5′UTR구유명현적절할작용，절할적위점재파서렬77~89 n t지간，속우특이성절할，구유포내항병독활성。결론구건료일충대HCV 5′UTR구유파향절할활성적M1GS핵매，차구유포내항병독활성，위동물모형내항병독효응적평개제공료실험재료，위신형항HCV약물적연구전정료기출。
Objective?To construct a targeting ribozyme (M1GS) which is specific to HCV genome. Methods According to the sequence of conservative region (5′UTR) of HCV genome, a guide sequence was designed and synthesized. And then by the PCR method, a kind of targeting ribozyme can be constructed by covalently linking the guide sequence to the 3′terminus ofM1 RNA, the catalytic subunit of Rnase P from Escherichia coli. Results The engineered ribozyme (M1GS-HCV/C76 is targeted to the 5′UTR of HCV genome, and can effectively cleave the substrate RNA segment in vitro. The cleavage is specific and the cleavage site is between 77 nt and 88 nt of the target region. Conclusion The M1GS-HCV/C76 would be a useful experimental material to further study its cleavage activity in vivo, and can be even used for evaluating its anti-viral effect in the animal model It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications and lay the foundation for developing a new nucleic acid drug of anti-HCV therapy.