目的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性产妇乳汁中含有乙型肝炎病毒(hepatitis B virus,HBV),但母乳喂养不增加母婴传播风险。既往研究显示母乳具有与HBsAg结合的特点,本研究旨在确定母乳中能与HBsAg结合的成分...目的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性产妇乳汁中含有乙型肝炎病毒(hepatitis B virus,HBV),但母乳喂养不增加母婴传播风险。既往研究显示母乳具有与HBsAg结合的特点,本研究旨在确定母乳中能与HBsAg结合的成分。方法本研究于2015年6月至2017年2月在南京大学医学院附属鼓楼医院完成。取2例HBV血清学标志均阴性的健康产妇的乳汁,同时用牛乳和山羊乳为对照。通过间接免疫印迹技术,观察高纯度重组酵母HBsAg与乳清蛋白结合情况,结合蛋白质谱分析结果,进一步通过竞争抑制实验,确定母乳中能与HBsAg结合的蛋白成分。结果间接免疫印迹显示,2例母乳乳清泳道均可在分子量约80 000处发现明显的反应条带,而牛乳和羊乳无相应条带。该反应条带处的蛋白质经蛋白质谱分析,提示与5种蛋白质序列同源性为28.4%~93.4%,其中同源性最高(93.4%)的蛋白为人乳铁蛋白。使用纯化的重组人乳铁蛋白,进一步经间接免疫印迹验证,显示人乳铁蛋白能与重组HBsAg结合。竞争抑制实验证明,纯化的重组人乳铁蛋白能显著抑制HBsAg与乙肝表面抗体的结合,且随乳铁蛋白浓度降低,抑制率降低。结论母乳中人乳铁蛋白能与HBV结合。进一步研究乳铁蛋白能否抑制HBV的感染性,对阐明母乳喂养不增加HBV母婴传播具有重要意义。展开更多
为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TAL...为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。展开更多
[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer techn...[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.展开更多
文摘目的乙型肝炎表面抗原(hepatitis B surface antigen,HBsAg)阳性产妇乳汁中含有乙型肝炎病毒(hepatitis B virus,HBV),但母乳喂养不增加母婴传播风险。既往研究显示母乳具有与HBsAg结合的特点,本研究旨在确定母乳中能与HBsAg结合的成分。方法本研究于2015年6月至2017年2月在南京大学医学院附属鼓楼医院完成。取2例HBV血清学标志均阴性的健康产妇的乳汁,同时用牛乳和山羊乳为对照。通过间接免疫印迹技术,观察高纯度重组酵母HBsAg与乳清蛋白结合情况,结合蛋白质谱分析结果,进一步通过竞争抑制实验,确定母乳中能与HBsAg结合的蛋白成分。结果间接免疫印迹显示,2例母乳乳清泳道均可在分子量约80 000处发现明显的反应条带,而牛乳和羊乳无相应条带。该反应条带处的蛋白质经蛋白质谱分析,提示与5种蛋白质序列同源性为28.4%~93.4%,其中同源性最高(93.4%)的蛋白为人乳铁蛋白。使用纯化的重组人乳铁蛋白,进一步经间接免疫印迹验证,显示人乳铁蛋白能与重组HBsAg结合。竞争抑制实验证明,纯化的重组人乳铁蛋白能显著抑制HBsAg与乙肝表面抗体的结合,且随乳铁蛋白浓度降低,抑制率降低。结论母乳中人乳铁蛋白能与HBV结合。进一步研究乳铁蛋白能否抑制HBV的感染性,对阐明母乳喂养不增加HBV母婴传播具有重要意义。
文摘为了敲除山羊乳中致敏源β-乳球蛋白(BLG)基因,同时在BLG基因座定点整合人乳铁蛋白(hLF)基因。首先针对山羊BLG第3外显子识别位点设计了1对特异性TALEN-3-L/R质粒对;同时,构建了含有1个HSV-TK负筛选基因的hLF基因打靶载体BLC14-TK。TALENs质粒对转染山羊胎儿成纤维细胞,2μg/m L嘌呤霉素筛选3 d,PCR扩增产物测序来验证其切割DNA活性。打靶载体BLC14-TK与TALEN-3-L/R质粒对共转染山羊胎儿成纤维细胞,经700μg/m L G418和2μg/m L GCV共筛选药物抗性细胞株;通过整合检测和同源重组检测来筛选hLF基因打靶细胞株;BLG~–/hLF^+打靶细胞株作为供核细胞进行山羊体细胞核移植。结果为:TALEN-3-L/R致突变率为25%-30%;获得BLG~–/hLF^+打靶细胞6株;共制作重构胚胎335枚,移植受体山羊23只,B超检测到30-35 d的妊娠受体9只(妊娠率39.1%),其中1只50日龄克隆胎儿验证为BLG~–/hLF^+基因型。以上结果表明获得BLG基因座定点整合hLF基因的基因打靶山羊是可行的,为培育羊乳中含低致敏原和富含hLF的山羊新品系奠定了基础。
基金Supported by the National High-tech R&D Program(2004AA213072)the Doctor Fund of Henan University of Science and Technology~~
文摘[Objective] The aim of this study is to understand the effects of donor cell type,embryo stage,number and transfer position on the efficiency of goat transgenic clone.[Method] Using somatic cell nuclear transfer technology,the single goat fetal fibroblasts(GFF)and mammary gland epithelial cells(GMGE)harboring human lactoferrin(hLF)gene were transferred to the enucleated oocyte.Reconstructed karyoplast-cytoplast couplets were fused,activated,and cultured in vitro.Embryos at 2-8 cell stage were transferred into oviduct of synchronized recipients,and blastocysts were transferred into uterine horn.[Result] The pregnancy rate was similar between GFF and GMGE(oviduct transfer:26.47% vs.20.00%),and between oviduct transfer and uterine horn transfer(26.47% vs.25.00%)for GFF group;pregnancy rate in the group with the mean number of embryo transferred per recipient of 21.2 was significantly higher than in those the 5.93 group and 9.64 group(40.00% vs.26.67% and 21.43%).[Conclusion] These results indicate that pregnancy rate of goat transgenic clone couldn't be affected by donor cell type,embryo stage and transfer position but be done by the number of embryo transferred per recipient.In addition,the study also suggests the feasibility of making transgenic goat using GMGE as donor cells.
