非缺失型α地中海贫血基因的克隆化分离

从一例基因型为αα~T/—的血红蛋白H病患者的手术切除脾制备了染色体DNA,用BamHⅠ水解后,回收14±1kb的DNA片段,作为插入体。以λEMBL_4噬菌体经非超离心法制备基因组DNA作为替换载体。经BamHⅠ水解回收左右臂,与α地贫脾DNA 14kb片段连接,包装、传染后,得10~4—10~5重组噬菌体/μg。以人α珠蛋白基因特异的探针作Benton原位杂交,M4×10~3克隆中筛出两个含α珠蛋白基因序列的克隆株。其中一株扩增后制备的重组DNA经酶解图谱及Southern印迹杂交鉴定,中间可替换部分含有14kb的人α珠蛋白基因组DNA。从而在我国首次从中国地贫病人中,以基因工程的方法,分离了克隆化的非缺失型α地贫基因,为研究其结构与功能打下了重要基础。

ISOLATION AND CLONING OF A NON-DELETION a- THALASSEMIA GENE

The Chromosomal DNA,prepared from the splenectomic sample of a patient suffered from hemoglobin H disease with genotype αaT/--was digested by BamHI,and the 14±1kb fragments were recovered and used as the insert.Meanwhile,the vector λEMBL4 DNA,prepared by a non-ultra centrifugation procedure,was digested by the same enzyme and the two arms were recovered for recombination.After Ligation,in vitro packaging,and tranfection,recombinants of 104-105 clones/μg DNA were obtained.With Benton hybridization technique in situ,two positive plaques were selected out of 4×103 clones as detected by an α-globin specific probe.One recombinant DNA was identified by restriction mapping and southern blots.The results showed that 14kb of a-globin genomic DNA had been inserted into this recombinant in place of the stuffer λDNA.This was the first time we succeeded in isolation of a cloned nondeletion α-thalassemia gene from a Chinese patient.