Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alter...Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 7612 to 15928 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.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
文摘Butyrylcholinesterase(BCHE)is a natural bioscavenger that protects humans against organophosphate toxicity.Due to the limited yield of human BCHE(hBCHE)when purifying from human plasma,it is necessary to find an alternative method to produce this protein.One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE.In this study,we cloned the hBCHEgene into a human lactoferrin(hLF)bacterial artificial chromosome(BAC)construct to make a hLFhBCHE BAC construct.Subsequently,we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice.Expression analysis showed that recombinant hBCHE(rhBCHE)was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 7612 to 15928 mg·L^(–1).Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE.Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.
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。
