摘要
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.
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.
基金
This work was supported by grant No.39730 46 0 of the National Nature Science F oundation
sup-ported by grant No.G19990 5 5 90 2 of National"973" Project
supported by National L aboratory ofContraceptives and Devices Research
