根据GenBank中纤连蛋白结合蛋白A基因(fnbA)序列设计了1对特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增;结果获得了3 600bp的DNA片段。将PCR产物克隆至pGEM T easy载体中,成功地构建了克隆质粒pGEM-fnbA。以HindⅢ和XhoⅠ...根据GenBank中纤连蛋白结合蛋白A基因(fnbA)序列设计了1对特异性引物,以金黄色葡萄球菌基因组DNA为模板,进行PCR扩增;结果获得了3 600bp的DNA片段。将PCR产物克隆至pGEM T easy载体中,成功地构建了克隆质粒pGEM-fnbA。以HindⅢ和XhoⅠ双酶切pGEM-fnbA和pET28a(+),将纯化的基因fnbA亚克隆至pET28a(+)中,构建了原核表达质粒pET28a-fnbA,并将其转化至E.coliBL21(DE3)感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,在约165 ku处出现了与预期目的蛋白一致的外源蛋白带。Western-blotting分析表明,该蛋白具有金黄色葡萄球菌的抗原性。展开更多
[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturatio...[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.展开更多
文摘[Objective] The research aimed to enhance culture efficiencies of oocyte and embryo of goat in vitro and to explore serum-free culture system in vitro.[Method] At present,the conventional solutions of oocyte maturation and embryo development in vitro were always added into 1% ITS(Insulin-transferrin-selenium) or using 1% ITS to replace FBS in 2 kinds culture solutions for conducting in vitro cultures of goat oocyte and parthenogenetic embryo.The influences of ITS on their developments were detected.[Result] ITS in maturation liquid of oocytes could not increase oocytes maturation rate but significantly increased blastocyst rate (58.06% vs. 48.19%)of parthenogenetic embryo.If FBS in maturation liquid of oocytes was replaced by ITS, the maturation rate, cleavage rate and blastocyst rate were basically unchanged.Adding ITS into embryo medium could increase blastocyst rate (68.30% vs. 56.82%)of parthenogenetic embryo of goat.If FBS in embryo medium was replaced by ITS,the cleavage rate didn’t change basically,while the blastocyst rate in ITS was obviously lower than that in FBS group(42.33% vs.56.82%).[Conclusion] ITS could promote maturation of oocyte in vitro and early embryonic development, in addition,ITS could replace serum in maturation medium of oocyte as serum-free culture system for conducting relevant researches.
