鸡PTH基因克隆、序列测定及其真核表达质粒的构建

Gene Cloning,Sequencing and Eukaryotic Expression Plasmid Construction of Chicken Parathyroid Hormone

采集200日龄产蛋期新扬州鸡甲状旁腺组织,提取RNA并进行反转录聚合酶链反应(RT-PCR),扩增产物进行T-A克隆、测序。序列测定结果表明:PTH cDNA核苷酸长度为360bp。与GenBank上发表的鸡序列比较,核苷酸同源性为99.6%,在第159位处存在C→A的有意义突变。与从GenBank下载读取的牛、马、人、狗、猫、食蟹猴PTH基因序列进行比较分析,同源性分别为51.4%、50.8%、50.6%、50.3%、50.3%和46.9%。将该基因片段克隆到真核表达载体pCEP4,构建重组质粒pCEP4-PTH,所获重组质粒经过酶切、测序鉴定,证实含有目的片段,且连接、构建正确,为利用pCEP4-PTH调控机体钙代谢、改善蛋鸡生产性能和蛋壳质量的研究应用奠定了基础。

One pair of primers were designed according to chicken PTH gene sequence published in GenBank. Chicken PTH gene was amplified by RT-PCR from Chicken parathyroidenm total RNA extracts. The result showed that the full length of Chicken PTH gene was consisted of 360 bp. The homology of the nueleotides compared to known sequence was 99.6%, since one nucleotide changed from C to A at 159 baseoThe homology of the nucleotide compared to toad, cattle, horse, people, dog, eat and CEM were 51.4%, 50.8%, 50.6%, 50.3%, 50.3%, 46.9% respectively. Then, the PTH was sub cloned into the eukaryotic expression vector pCEP4 and such recombinant plasmid was transformed into the host E. colt strain DH5α for identification. The result showed that the recombinant plasmid pCEP4-PTH was constructed correctly, which paved the way of utilizing pCEP4-PTH to regulate calcium metabolism, improve laying performance and research the eggshell quality.