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
采用RT-PCR技术扩增禽呼肠孤病毒(ARV)S1133毒株和广西分离株R 1的2σ基因。将2σ基因克隆至PGEX-4T-1载体上,测序结果表明,插入的片段为2σ目的基因。切下目的基因2σ重组到含有谷胱甘肽(G ST)的融合蛋白质原核表达载体PGEX-4T-1,获得...采用RT-PCR技术扩增禽呼肠孤病毒(ARV)S1133毒株和广西分离株R 1的2σ基因。将2σ基因克隆至PGEX-4T-1载体上,测序结果表明,插入的片段为2σ目的基因。切下目的基因2σ重组到含有谷胱甘肽(G ST)的融合蛋白质原核表达载体PGEX-4T-1,获得重组质粒。经PCR、酶切以及序列分析鉴定,表达2σ基因插入的位置、大小和读码框架均正确,表明成功构建了融合表达载体PGEX-S1133-2σ和PGEX-R 1-2σ。构建好的重组质粒,在大肠杆菌JM 109α中经1 mm o l/L IPTG诱导得到了表达。融合蛋白G ST-2σ和G ST R 1-2σ的相对分子质量为65 100,以包涵体形式存在。W estern-b lot分析表明,融合蛋白能够与ARV阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。展开更多
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.
文摘采用RT-PCR技术扩增禽呼肠孤病毒(ARV)S1133毒株和广西分离株R 1的2σ基因。将2σ基因克隆至PGEX-4T-1载体上,测序结果表明,插入的片段为2σ目的基因。切下目的基因2σ重组到含有谷胱甘肽(G ST)的融合蛋白质原核表达载体PGEX-4T-1,获得重组质粒。经PCR、酶切以及序列分析鉴定,表达2σ基因插入的位置、大小和读码框架均正确,表明成功构建了融合表达载体PGEX-S1133-2σ和PGEX-R 1-2σ。构建好的重组质粒,在大肠杆菌JM 109α中经1 mm o l/L IPTG诱导得到了表达。融合蛋白G ST-2σ和G ST R 1-2σ的相对分子质量为65 100,以包涵体形式存在。W estern-b lot分析表明,融合蛋白能够与ARV阳性血清发生特异性反应,表明该重组蛋白具有良好的反应原性。
