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目的 :构建特异性切割人组织金属蛋白酶抑制剂 1 (tissue inhibitor of m etalloproteinases1 ,TIMP- 1 )的锤头状核酶的真核表达载体并在体外进行活性鉴定 ,为应用于瘢痕基因治疗奠定基础。方法 :设计并合成针对人组织 TIMP- 1 m RNA的锤头状核酶基因 Rz1 82、Rz35 8和 Rz4 1 2及相应的点突变核酶基因 ,将核酶基因克隆于可在体内高表达核酶的载体 p BSK-neo U 6中 ,制备嵌合于 U 6 sn RNA分子的核酶基因克隆。逆转录聚合酶链式反应获得全长 TIMP- 1 m RNA基因片段并克隆至T载体。体外转录法大量制备以 α- 32 P U TP标记的核酶及靶 RNA,进行体外切割实验。 结果 :核酶基因克隆制备正确 ,在体外成功转录出嵌合于 U6 sn RNA的核酶和靶 RNA。 37℃的生理温度下 ,U6 Rz1 82和 U6 Rz35 8成功切割了靶 RNA,U6 Rz1 82切割效率为 4 9.2 3% ,Km=2 9.7nmol/ L ,Kcat=0 .32 m in- 1 。 U 6 Rz35 8切割效率为 5 5 .2 1 %。 Km=39.6 nm ol/ L ,Kcat=0 .2 1min- 1。U6 Rz4 1 2及突变核酶均未显示切割活性。结论 :本研究中制备的 U6 Rz1 82和 U6 Rz35 8有良好的特异催化切割活性 ,有望在瘢痕成纤维细胞内抑制人 TIMP- 1的表达 ,成为新的抗瘢痕核酸药物
OBJECTIVE: To construct an eukaryotic expression vector of hammerhead ribozyme that specifically cleaves tissue inhibitor of m-etalloproteinases 1 (TIMP-1) and to identify its activity in vitro, so as to lay the foundation for the gene therapy of scar . Methods: The hammerhead ribozyme gene Rz1 82, Rz35 8 and Rz4 1 2 and the corresponding point mutation ribozyme gene targeting TIMP-1 m RNA of human tissue were designed and synthesized. The ribozyme gene was cloned in the highly expressed core The enzyme vector p BSK-neo U 6 was used to prepare ribozyme gene clones chimeric to U 6 sn RNA molecules. The full-length TIMP-1 m RNA gene fragment was obtained by reverse transcription polymerase chain reaction and cloned into the T vector. In vitro transcription was used to prepare a large number of ribozymes labeled with α-32P U TP and target RNA for in vitro cleavage experiments. Results: The ribozyme gene clone was prepared correctly and the ribozyme and target RNA chimeric to U6 sn RNA were successfully transcribed in vitro. The target RNA was cleaved successfully at U6 Rz1 82 and U6 Rz35 8 at a physiological temperature of 37 ° C. The cleavage efficiency of U6 Rz1 82 was 4 9.23%, Km = 2 9.7 nmol / L, Kcat = 0.32 m in- 1. U 6 Rz35 8 Cutting efficiency of 5 5 .21%. Km = 39.6 nm ol / L, Kcat = 0.2 1 min-1. U6 Rz4 1 2 and mutant ribozyme showed no cleavage activity. CONCLUSION: The U6 Rz1 82 and U6 Rz35 8 prepared in this study have good specific catalytic cleavage activity and are expected to inhibit the expression of human TIMP-1 in scar fibroblasts and become a new anti-scarring nucleic acid drug