人ECK基因外显子3的实验研究

目的　建立人eck基因外显子 3(exon 3)的克隆与鉴定方法 ,研究其在ZR 75 1细胞系中的突变情况。方法　设计一对eck基因exon 3特异性引物 ,提取人正常皮肤组织和乳腺癌细胞系ZR 75 1基因组DNA ,并以此作为模板 ,采用聚合酶链反应 (PCR)技术扩增eck基因exon 3片段 ,克隆入中介载体pUCm T中构建重组质粒 ,转化JM10 9大肠杆菌 ,扩增后经酶切、PCR初步鉴定后 ,进行序列分析。结果　①从正常皮肤组织上皮细胞、ZR 75 1细胞系基因组DNA中 ,经PCR扩增 ,获得了人eck基因exon 3片段 ;②建立了正常皮肤组织、ZR 75 1细胞系eck基因exon 3片段的克隆 ;③ZR 75 1细胞系中eck基因exon 3片段存在突变。结论　从人组织与细胞系基因组DNA中 ,成功地构建了人类eck基因exon 3的克隆 ,并证实eck基因exon 3在ZR 75 1乳腺癌细胞系中有突变 ,为进一步研究eck基因exon 3在肿瘤形成中的作用奠定了基础

Experimental studies on human exon 3 of eck gene

Objective To establish a method of cloning and identifying the exon 3 of eck gene from normal tissue and ZR-75-1 cell line (a human breast cancer cell line) and study the mutation in these genes. Methods We designed a pair of primers specific to exon-3 of eck gene and amplified the exon 3 fragments from the extracted genomic DNAs derived from normal epithelial cells of skin tissue and ZR-75-1 cells respectively using PCR technique. The E.coil JM109 was transformed with recombinant plasmids constructed by inserting the amplified fragments into medium vector pUCm-T and these amplified fragments were sequenced after primary screening of endonuclease restriction digestion and PCR amplification and compared with that reported in GenBank. Results ① We obtained the genomic DNAs from human normal epithelial cells and ZR-75-1 cell line and amplified the fragments of human exon 3 of eck gene through PCR technique with the extracted DNAs as templates respectively. ② We obtained the cloning vectors of exon 3 of eck gene of human normal epithelial cells and ZR-75-1 cell line respectively. ③ Mutation of nucleotides was observed in ZR-75-1 cells. Conclusion We successfully established the method of cloning the human exon 3 of eck gene and found some mutations in the detected samples. This study lays a foundation for further study on the function of eck gene in tumorigenesis.