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
Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb D...Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb DPF 1 cDNA was obtained. Linear carrier pGEX 4T 3 was obtained by SmaI NotI digestion. The recombinant pGEX 4T 3/DPF 1 cDNA was constructed by ligating linear pGEX 4T 3 with 1.8 kb DPF 1 cDNA. Large quantity of fusion protein was expressed in E.coli DE3 induced by IPTG at 37℃. Cells were lysed by using lysozyme and the fusion proteins were purified. To induce anti DPF 1 antiserum, male BALB/c mice were immunized by using GST DPF 1 fusion protein. Conditioned cultured medium derived from rabbit oviduct mucosa epithelial cells was prepared and loss of function analysis of DPF 1 was done by adding anti DPF 1 antibodies in the conditioned medium co culture with mouse fertilized eggs. Result GST DPF 1 fusion protein was purified and antisera were prepared. After GST absorption, the antiserum shows high specificity to DPF 1. Anti DPF 1 antiserum absorbed with GST could totally inhibit the early embryos development of mouse cultured in the conditioned medium and there was a positive relation between the ratio of early embryos of mouse blocked at 2 cell stage and the dose of anti DPF 1 antiserum added to the conditioned medium. Conclusion 'Loss of function' analysis revealed that rabbit oviductin ' DPF 1'has the function of overcoming the early embryo development block.展开更多
DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on...DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on embryonic development and implantation. The results of experiment I indicated that of the 82 fertilized eggs cul-tured in the absence of DPF-1, only 9 were normally divided to the morula stage onday4. By contrast, in the medium containing DPF-1 109(90% ) of 121 embryos weredeveloped to the blastocyst stage. In eoperiment Ⅱ, the developed blastocysts weretransferred into the uterus of 3-day pseudopregnant DPF-1-treated or control recipi-ents. Three different combinations of intrauterine transfer were performed: the DPF-1 treated embryos transferred to the control reclpients(DPF- 1→C),the control embryosto the DPF- 1 treated recipients(C→DPF- 1), and the control embryos to the control re-cipients (C→C). The implantation rate was assessed on day 18 after transfer. Therewere significant differences in pregnancy rate per mouse between DPF-1→C and C→DPF- 1 or control groups(C→C). The embryo implantation rate was low in C→DPF-1 (26. 0%, 75/288) and C→ C (25 3%, 73/288) as compared with DPF-1→C(61. 8%, 178/288). These results suggest that the major site of the DPF-1 actionduring early pregnancy is the embryo itself. DPF-1 enables embryos to develop, buthas no obvious direct effect on uterine receptivity.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This work was supported by grant No.39730 46 0 of the National Nature Science F oundationsup-ported by grant No.G19990 5 5 90 2 of National"973" Projectsupported by National L aboratory ofContraceptives and Devices Research
文摘Objective To reveal the function of rabbit oviductin DPF 1 on overcoming the early embryo development block Method 1.9 kb DPF 1 cDNA derived from recombinant pBluescript IIKS was digested by HincII, then 1.8 kb DPF 1 cDNA was obtained. Linear carrier pGEX 4T 3 was obtained by SmaI NotI digestion. The recombinant pGEX 4T 3/DPF 1 cDNA was constructed by ligating linear pGEX 4T 3 with 1.8 kb DPF 1 cDNA. Large quantity of fusion protein was expressed in E.coli DE3 induced by IPTG at 37℃. Cells were lysed by using lysozyme and the fusion proteins were purified. To induce anti DPF 1 antiserum, male BALB/c mice were immunized by using GST DPF 1 fusion protein. Conditioned cultured medium derived from rabbit oviduct mucosa epithelial cells was prepared and loss of function analysis of DPF 1 was done by adding anti DPF 1 antibodies in the conditioned medium co culture with mouse fertilized eggs. Result GST DPF 1 fusion protein was purified and antisera were prepared. After GST absorption, the antiserum shows high specificity to DPF 1. Anti DPF 1 antiserum absorbed with GST could totally inhibit the early embryos development of mouse cultured in the conditioned medium and there was a positive relation between the ratio of early embryos of mouse blocked at 2 cell stage and the dose of anti DPF 1 antiserum added to the conditioned medium. Conclusion 'Loss of function' analysis revealed that rabbit oviductin ' DPF 1'has the function of overcoming the early embryo development block.
文摘DeveloPment Promoting Factor-1 (DPF-1) was derived from the rabbit oviductmucosa epithelial cells. fertilized eggs were treated with DPF-1 and it was adminis-tered to female mice in order to investigate its effect on embryonic development and implantation. The results of experiment I indicated that of the 82 fertilized eggs cul-tured in the absence of DPF-1, only 9 were normally divided to the morula stage onday4. By contrast, in the medium containing DPF-1 109(90% ) of 121 embryos weredeveloped to the blastocyst stage. In eoperiment Ⅱ, the developed blastocysts weretransferred into the uterus of 3-day pseudopregnant DPF-1-treated or control recipi-ents. Three different combinations of intrauterine transfer were performed: the DPF-1 treated embryos transferred to the control reclpients(DPF- 1→C),the control embryosto the DPF- 1 treated recipients(C→DPF- 1), and the control embryos to the control re-cipients (C→C). The implantation rate was assessed on day 18 after transfer. Therewere significant differences in pregnancy rate per mouse between DPF-1→C and C→DPF- 1 or control groups(C→C). The embryo implantation rate was low in C→DPF-1 (26. 0%, 75/288) and C→ C (25 3%, 73/288) as compared with DPF-1→C(61. 8%, 178/288). These results suggest that the major site of the DPF-1 actionduring early pregnancy is the embryo itself. DPF-1 enables embryos to develop, buthas no obvious direct effect on uterine receptivity.
