Monoclonal antibodies (McAb) were prepared by fusing sp2/0 myeoma cell with spleen cell of mice Balb/c which were immunized with 64 kDa rabbit oviductin (DPF-1). Supernatants of hybridoma cell were screened for produc...Monoclonal antibodies (McAb) were prepared by fusing sp2/0 myeoma cell with spleen cell of mice Balb/c which were immunized with 64 kDa rabbit oviductin (DPF-1). Supernatants of hybridoma cell were screened for producing anti-DPF1 McAbs with enzyme-linked immunosorbent assay (ELISA) and Western blotting. Positive hybrids were cloned by using the technique of limiting dilution. Two strains of hybridoma cell were obtained,named 3E8 and 5A7. Both McAbs were determined as I'm. Results of Western blotting showed that the two McAbs recognised the DPF-1 hand (64kDa) specifically. By using McAb-5A7 as probe, we found that DPF-1 did not occur in the tissues of spleen, uterus, ovary,small intestine, skeleton muscle, brain, liver,heart, lung and kidney, except for that of oviduct; some DPF-1 homologous molecules were also revealed itself in that of oviduct tissues of mouse and golden hamster Their apparent molecular weights were 32kDa,72kDa in mouse, and 49kDa in golden hamster. All these results indicated that DPF-1 is tissue specific not species specific. (To whon all corrcspondcnce should be addrcssed. Prcsent addrcss Shanghai Institute of Planned Parenthood Rcscarch. Shanghai 200032. PRC. )展开更多
A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductin...A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mous e cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn’t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.展开更多
文摘Monoclonal antibodies (McAb) were prepared by fusing sp2/0 myeoma cell with spleen cell of mice Balb/c which were immunized with 64 kDa rabbit oviductin (DPF-1). Supernatants of hybridoma cell were screened for producing anti-DPF1 McAbs with enzyme-linked immunosorbent assay (ELISA) and Western blotting. Positive hybrids were cloned by using the technique of limiting dilution. Two strains of hybridoma cell were obtained,named 3E8 and 5A7. Both McAbs were determined as I'm. Results of Western blotting showed that the two McAbs recognised the DPF-1 hand (64kDa) specifically. By using McAb-5A7 as probe, we found that DPF-1 did not occur in the tissues of spleen, uterus, ovary,small intestine, skeleton muscle, brain, liver,heart, lung and kidney, except for that of oviduct; some DPF-1 homologous molecules were also revealed itself in that of oviduct tissues of mouse and golden hamster Their apparent molecular weights were 32kDa,72kDa in mouse, and 49kDa in golden hamster. All these results indicated that DPF-1 is tissue specific not species specific. (To whon all corrcspondcnce should be addrcssed. Prcsent addrcss Shanghai Institute of Planned Parenthood Rcscarch. Shanghai 200032. PRC. )
基金Supported by National Natural Science Foundation of China (39730460)National "973" Project (G1999055902)National Labora-
文摘A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mous e cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn’t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.
