银鲫种系细胞标记分子Vasa: cDNA克隆及其抗体制备

种系细胞始自胚胎发育早期,是动物生殖及生殖工程的基础。为研究鱼类的种系细胞提供标记分子,我们克隆并鉴定了银鲫的vasacDNA即Cagvasa。CagvasacDNA全长2771碱基(nt),编码的蛋白为银鲫Vasa即CagVasa,全长701个氨基酸(aa)。CagVasa蛋白与已知Vasa蛋白的结构特征一致:在N端有14个RGG重复序列,在C端Vasa所特有的8个功能域俱全。银鲫Vasa与鲤鱼、斑马鱼、陆生脊椎动物和果蝇的Vasa蛋白分别有95%,89%,61%-66%和50%的同源性。卵巢切片的RNA原位杂交揭示,Cagvasa限于种系细胞,且表达水平呈现出低-高-低的动态变化:即两头低(卵原细胞跟Ⅳ期成熟卵子),中间高(Ⅱ-Ⅲ期卵子)。为分析鱼类种系细胞提供手段,我们用310aa的N端序列产生细菌的重组蛋白来免疫大白兔,获得了抗Vasa的多克隆抗体αVasa。Western免疫印迹表明,αVasa特异性地识别一个鱼类性腺的蛋白,该蛋白的分子量为75kD,仅见于银鲫的性腺和卵子。卵巢切片的组织免疫荧光共聚焦显微分析表明,抗体αVasa只对种系细胞染色:卵原细胞着色最深,卵母细胞和早期的卵子都浓染,成熟卵则浅染。类似情况亦见之于精子发生早期阶段的雄性种系细胞。卵巢和精巢的体细胞则不着色。因此,Cagvasa编码的当是Vasa同源蛋白,为银鲫种系细胞的第一个标记分子。我们的研究表明,抗体αVasa染色灵敏度高,特异性好,当是鉴别银鲫及其它鲤科鱼类的种系细胞的有效手段

Gibel carp germ cell marker Vasa: cDNA cloning and its antibody preparation

The basis for animal reproduction and reproductive biotechnology is germ cells that are segregated from the somatic lineage early in embryonic development and produce sperm and eggs for germline transmission between generations. To provide a germ cell marker, we cloned and characterized Cagvasa, a vasa homolog from the gibel carp Carassius auratus gibelio. The Cagvasa cDNA is 2 771 nt (nucleotide) and encodes a 701-aa(amino acid) protein (CagVasa) that possesses 14 RGG-repeats and all the eight conserved motifs of Vasa proteins. CagVasa shows sequence identities of 95%, 89%, 61%-66% and 50% to Vasa proteins from the common carp, zebrafish, tetrapods and Drosophila, respectively. RNA in situ hybridization on ovarian sections showed that Cagvasa was absent in any gonadal somatic cells but dynamically expressed throughout oogenesis: the signal is weak in oogonia, peaks in vitellogenic oocytes and deceases in maturing oocytes. To develop a tool for analyzing fish germ cells, we generated αVasa, a rabbit antibody against a recombinant protein of 310-aa N-terminal CagVasa. Western blot analyses showed that αVasa detected a 75 kD protein exclusively in the gibel carp gonads and isolated oocytes. To confirm the germ cell-specifity of αVasa, fluorescent immunostaining was performed on gonadal sections. This antibody stains exclusively germ cells throughout oogenesis and early stages of spermatogenesis. We conclude that vasa encodes a Vasa ortholog that services a first germ cell marker in the gibel carp, and that αVasa provides a highly sensitive and powerful tool for the analysis of germ cell specification and differentiation in the gibel carp