绵羊Gfrα1基因的部分cDNA克隆与抗原表位多肽的原核表达

Cloning of Gfrα1 Partial cDNA in Sheep and Prokaryotic Expression of Epitope Polypeptide

旨在克隆绵羊Gfrα1基因序列,深入研究绵羊精原干细胞(SSCs)。本研究通过RT-PCR扩增和分子克隆的方法克隆到了绵羊Gfrα1基因的编码区大部分片段。结果,克隆得到的绵羊Gfrα1基因的大部分cDNA序列长1 312bp,包括1 311bp的开放阅读框(ORF),编码437个氨基酸,与牛、人、黑猩猩、大鼠和小鼠相应核苷酸序列的同源性分别为98.5%、91.3%、91.0%、88.9%和88.0%。根据获得的cDNA序列,预测已知片段的抗原表位区,并将抗原表位区序列插入pET-44a(+)载体,经转化得到重组菌,经IPTG诱导表达,并通过亲和柱层析,纯化制备GFRα1抗原表位多肽。Western blot检测发现,经诱导表达的GFRα1抗原表位多肽约为18.2ku,与预测的大小一致。绵羊Gfrα1基因的克隆和表达研究,为进一步制作该基因的多克隆抗体奠定基础,为绵羊SSCs的分子水平鉴定及功能分析提供了研究条件。

In order to clone the DNA sequence of Gfral gene in sheep and deeply research the sheep spermatogonial stem ceils （SSCs）, in the present study, the partial sheep Gfral cDNA were cloned using RT-PCR and molecular cloning technique. The cloned partial Gfral gene was 1 312 bp in length, including an ORF of 1 311 bp encoding 437 amino acids. The nucleotide sequences of sheep Gfral gene were compared with the counterpart sequences of Bos Taurus, homo sapiens, Pongo abelii , Rattus norvegicus and Mus musculus , and the nucleotide homology was 98.5%, 91.3%, 91.0%, 88.9% and 88.0%, respectively. According to the cloned Gfral gene sequence, the epitope was forecasted and then the eDNA was inserted into pET-44a（＋） vector. Subsequently, the recombinant was induced by IPTG. The recombinant epitope polypep- tide was purified according to Affinity column chromatography. Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate 18.2 ku. The data collected provided the important information for further making Gfral gene polyelonal antibody and authenticating on molecular level for spermatogonial stem cells in sheep.