[Cloning and identification of recombinant cDNA to a rabbit oviductin "DPF-1"].
A recombinant cDNA library to polyA + RNA isolated from rabbit oviduct epithelial cells was constructed, and screened with a polyclonal antibody against DPF-1 (64 kDa). 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-Not1 digestion method, the insert cDNA fragment size of these three recombinants was revealed to be 0.8 kb, 1.2 kb and 1.2 kb respectively (Fig. 3). These cDNA fragments were then isolated and subcloned into pBluescriptKS, and recombinant plasmids (pDPF-1.1, pDPF-1.2 and pDPF-1.3) were constructed (Fig. 4). Dot blot hybridization with a 32p-labeled 1.2 Kb-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).
Shanghai Institute of Cell Biology, Chinese Academy of Sciences.
C J
CJ
Liu
H
H
Shen
Z
Z
Gu
J N
JN
Lu
G X
GX
Cheng
J K
JK
Tso
D000818:Animals; D003001:Cloning, Molecular; D004847:Epithelial Cells; D005187:Fallopian Tubes / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D011817:Rabbits; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*
D012697:Serine Endopeptidases; C074599:oviductin