猪骨形态发生蛋白4成熟肽cDNA的克隆及序列分析

克隆猪骨形态发生蛋白4(BMP-4)成熟肽基因。通过提取猪肾的总RNA,通过RT-PCR技术扩增出目的基因,并将其回收后与pMD18-T载体连接,转化大肠杆菌TOP10感受态细胞,进行阳性克隆的筛选与鉴定。琼脂糖凝胶电泳检测RT-PCR产物,显示结果与预期片段大小一致,质粒PCR检测、酶切鉴定证实重组克隆中插入了PCR产物,测序结果揭示BMP-4成熟肽cDNA长为351bp,编码116个氨基酸。序列对比表明,猪与人、小鼠、牛、绵羊BMP-4成熟肽cDNA序列的同源性分别为94.87%、90.60%、94.87%、94.59%,而相应的氨基酸同源性分别为99.12%,99.12%,100%、100%。首次成功地克隆了猪BMP-4成熟肽编码基因,对于进一步研究猪BMP-4全基因结构、功能及其与繁殖性能的关系有重要的意义。

Cloning and Sequencing of the mature peptide cDNA of bone morphogenetic protein-4 gene in pig

To clone the cDNA of sus scrafa mature peptide of bone morphogenetic protein-4.Totle RNA was extracted from kidney of sus scrafa and the desired segment was obtained by RT-PCR with two specific primers.The PCR products was cloned into pMD18-T vector and transformed into Top-10 competent cells.After PCR and double enzyme screening,the sequence of insert in the recombinant plasmid was assayed by DNA sequence machine.DNA agarose electrophoresis showed that the products of RT-PCR was consistent with desired BMP-4.PCR,double enzyme digestion revealed that the PCR product was inserted pMD18-T vector and sequencing demonstrated that the mature protein cds of pig BMP-4 was consisted of 351 nucleotides and encoded 116 amino acids.Sequence alignments showed that the mature protein cds of pig BMP-4 had 94.87%,90.60%,94.87% and 94.59% homologies with that of human,mouse,cow,and sheep respectively,while the homologies for the deduced amino acids were 99.12%,99.12%,100% and100% respectively.It is concluded that the mature protein cds of pig BMP-4 have been successively cloned,and this will promote the further studies on the full gene structure and function of this gene as well as its relationship with pig reproduction.