A study of the molecular mechanism and the gene therapy for congenital myotonic dystrophy.

1、先天性强直性肌营养不良的产前诊断方法：建立了非放射性同位素聚合酶链式反应和Southern印迹杂交方法，分析肌钙蛋白/蛋白激酶基因的CTG重复大小异常对该患者的诊断价值。对该基因的CTG重复序列大小和侧翼标记进行分析，用于产前诊断。这些方法主要用于分析脆性X综合征和亨廷顿病的基因异常，基因异常的机制相似。2、分析该病淋巴母细胞中肌钙蛋白/蛋白激酶基因的表达。建立竞争性聚合酶链式反应技术，比较正常和先天性强直性肌营养不良患者的基因表达水平。肌钙蛋白/蛋白激酶基因在正常组织和疾病组织中的表达水平无明显差异。在淋巴母细胞中，肌钙蛋白/蛋白激酶基因的信使核糖核酸含量很低。3、肌钙蛋白/蛋白激酶蛋白在大肠杆菌中的表达。我们尝试利用大肠杆菌表达系统来制备肌钙蛋白/蛋白激酶蛋白。从人和小鼠中制备了肌钙蛋白/蛋白激酶的全长cDNA，并将其亚克隆到pRSET载体上，在大肠杆菌中进行了表达。肌钙蛋白/蛋白激酶蛋白在该体系中检测不清。我们将尝试使用该基因的部分片段或使用其他表达系统。

1, An method of the prenatal diagnosis for congenital myotonic dystrophy.We established the non-radioisotope PCR and Southern blot method to analyze CTG repeat size abnormality of the myotonin/protein kinase gene for the diagnosis of the patient. The CTG repeat size and flanking markers of this gene were analyzed for the prenatal diagnosis. Principally, these methods were also used for the analysis the gene abnormality of fragile X syndrome and Huntington disease in which the mechanism of the gene abnormality are similar.2, Analysis of the myotonin/protein kinase gene expression in the lymphoblasts of the disease.We analyzed the myotonin/protein kinase mRNA in lymphoblasts by a PCR technology. A competitive PCR technology was developed to compare the amount of mRNA between the normal and the congenital myotonic dystrophy samples. The expression level of the myotonin/protein kinase gene was not different in the normal and the disease samples. The mRNA amount of the myotonin/protein kinase gene was very low in the lymphoblasts. We will try to use the muscle for this study.3, Expression of the myotonin/protein kinase protein in the E.coli.We tried to make the myotonin/protein kinase protein by a E.coli expression system. The full length myotonin/protein kinase cDNAs were prepared from the human and mouse, and these cDNAs were subcloned into pRSET vectors in the system of E.coli expression. The myotonin/protein kinase protein was not detected in this system clearly. We will try to use the partial fragment of the gene or use other expression system.