人生长分化因子-5完整成熟肽基因的克隆

The cloning of integral human GDF-5 mature peptide gene

目的 :克隆人生长分化因子 5 (hGDF 5 )完整成熟肽基因。方法 :根据Genbank中hGDF 5的序列化学合成两条引物 ,从人胎儿软骨组织提取总RNA ,通过反转录聚合酶链式反应 (RT PCR )得到hGDF 5完整成熟肽基因。将所得基因片段插入克隆载体 pMD18 T并转化大肠杆菌DH5α ,提取重组质粒 ,酶切鉴定并测序。结果 :DNA琼脂糖凝胶电泳显示 :PCR产物为一长约 3 80bp的带 ,阳性克隆质粒经双酶切可切出约 3 80bp的片段。全自动DNA测序表明与Genbank中的序列完全相符。 结论 :通过反转录聚合酶链式反应从人胎儿软骨组织中成功克隆出人GDF 5完整成熟肽基因 ,基因序列完全正确。

Objective: To clone integral human growth differentiation factor-5(GDF-5) mature peptide gene. Methods: Two primers were chemosynthesized according to the hGDF-5 sequence reported in Genbank. The hGDF-5 gene was gained by RT-PCR from the cDNA which was extracted from human fetus cartilage tissue, and was cloned into vector pMD18-T. The sequence of recombinant plasmid pMD18-T- hGDF-5 was analyzed by sequence analysis. Results: DNA agarose gel electrophoresis showed that the product of RT-PCR was about 380bp, and double enzyme digestion of the recombinant plasmid corresponded with it. The result of sequence assay was in agreement with the reported hGDF-5 sequence in Genbank. Conclusion: The integral human GDF-5 mature peptide gene was cloned successfully, and totally identified with the sequence of human GDF-5 in Genbank.