摘要
以兔输卵管上皮细胞的polyA^+RNA为模板反转录合成cDNA,并构建了重组cDNA文库。库容量为2×10~6pfu/ml,重组体与非重组体比例为1000:1。以制备的豚鼠抗兔DPF-1(64 kDa)多克隆抗体克隆筛选到4个免疫阳性重组体(DPF-1.1、DPF-1.2、DPF-1.3和DPF-1.4),并被纯化。表位选择结果显示:DPF-1.1、DPF-1.2和DPF-1.3选择吸附的抗体可识别DPF-1和分子量为44 kDa的蛋白质。DPF-1.1、DPF-1.2和DPF-1.3经EcoRⅠ-NotⅠ双酶切后,得到的插入片段的长度分别为0.8 kb,1.2 kb,1.2 kb;纯化后亚克隆进p^(Bluescript)KS质粒,构建成重组质粒p^(DPF-1.1)、p^(DPF-1.2)和p^(DPF-1.3)。杂交结果表明,这些重组质粒可交叉杂交,说明它们拥有共同的核酸序列。点和Northern印迹分析结果显示,编码DPF-1的基因仅在输卵管组织中表达,具明显的组织专一性,且polyA+RNA的长度约为2.1 kb。本研究为揭示DPF-1的分子结构、功能及表达调控的机制奠定了基础。同时亦可望最终证实DPF-1的确切生物学作用。
A recombinant cDNA library to po-lyA+RNA isolated from rabbit oviduct epithelial cells was constructed, and screened with a polyclonal antibody against DPF-1 (64kDa). 4 immunopositive plaques (DPF-1.1, DPF-1.2, DPF-1.3 and DPF-1.4) were purified. The polyclonal antibodies were epitope-selected respectively against the fused proteins produced by these positive recombinant plaques. Identification of recombinant clones by epitope selection revealed that the epitope -selected antibodies from DPF-1.1, DPF-1.2 and DPF-1. 3 could recognise not only DPF-1, but 44 kDa protein also (Fig. 2). By using EcoRI-Notl digestion method, the insert cDNA fragment size of these three recombinants was revealed
to be 0.8kb, 1.2kb and 1.2kb respectively (Fig. 3). These cDNA fragments were then isolated and subcloned into pBiuescriptKS, and recombinant plasmids
(pDPF-1.1, pDPF-1.2 and pDPF-1.3) were con-
structed (Fig.4). Dot blot hybridization with a 32p-labeled 1.2Kb-insert of cDNA from pDPF-1.3 indicated that these recombinant plasmids could cross-hybridized (Fig. 5), further indicating that they all possessed a common nucleic acid sequence. Dot and Northern blotting analysis of total RNA prepared from eight different tissues (skeleton muscle, heart, kidney, oviduct, liver, spleen, lung and small intestine) showed that the gene encoding DPF-1 was expressed specifically in the oviduct tissue (Fig. 6, Fig. 7).
出处
《实验生物学报》
CSCD
1996年第4期395-401,共7页
Acta Biologiae Experimentalis Sinica
基金
国家自然科学基金~~
关键词
兔
输卵管因子
DPF-1
CDNA
Oviductin. DPF-1. Clone of cDNA. Developmental block of early embryo.
