抗丙型肝炎病毒锤头结构核酶的计算机设计和载体的构建

目的选择针对丙型肝炎病毒（HCV）5'非编码区（NCR）和核心区（C）的核酶（ribozyme，Rz）切割位点，构建带自剪切的Rz真核表达载体。方法应用计算机辅助设计，根据能量最低化原理，以HCV－H（1a）株SNCR和C靶序列，预测其二级结构，选择理想的Rz切割位点，设计锤头结构核酶；体外合成针对HCV5'NCR的Rz213和Rz260的cDNA，通过亚克隆技术连接于真核表达载体（pcDNA3）。结果在124个自然切割位点（CUX和GUX）中选出213（CUC）、260（GUA）、407（GUC）和498（CUU）4个切点；Rz213和Rz260的DNA序列分析，结果与合成序列完全一致，酶切鉴定两Rz连接正确。结论计算机可作为抗病毒Rz设计的重要辅助工具；通过亚克隆技术可使目的Rz两端带自剪切Rz，并成功地插入真核表达载体。

Computer Designation and construction of eukaryotic expression vector of the hammerhead ribozymes against hepatitis C virus

Objective To choose ideal cleavage sites and construct eukaryotic expression vector of the ribozymes (Rz) which can specifically cleave 5' noncoding region (5'NCR) and core (C) region of hepatitis C virus (HCV). Methods The secondary structure of 5'NCR and C region of HCV-H strain as target sequences was calculated with the help of computer, and the ribozymes against HCV were designed according to the "hammerhead structure" suggested by Symons; The cDNA of two ribozymes (213 and260) against the 5'NCR were synthesized chemically, and then were recombined in eukaryotic expression vector pcDNA3. Results 4 ribozymes directed 4 sites of HCV 5'NCR (213 and 260) and C region (470 and 498) were selected from 124 natural cleavage sites of hammerhead ribozymes. The sequencing for two ribozymes showed there was no difference between the synthetic sequences and the analytic sequences, andthey were appropriately cloned in pcDNA3 eukaryote vector by the enzyme-cutting identification of them. Conclusion The ideal cleavage sites of ribozymes aganinst HCV may be designed by the help of computer. The eukaryotic vectors of ribozymes with ets ribozymes were appropriately Inserted by the subclonal technique for DNA. They make the further in vivo study against HCV gene therapy available.