产生乳腺定位表达人CD14的转基因小鼠研究

Study on Production of Transgenic Mice Expressing Human CD14 in Mammary Gland

将来自ＰＣＲ法合成的绵羊β－乳球蛋白基因（ＢＬＧ）５′端８９８ｂｐ上游区和人单核细胞表面分化抗原基因（ｈＣＤ１４）成熟肽１２４５ｂｐ片段构建的ＢＬＧ－ｈＣＤ１４融合基因纯化后，注入小鼠受精卵原核．实验共注射受精卵１１４９枚，经体外培养发育到２－细胞期的胚胎计９６８枚，发育率为８４．２％．从发育的胚胎中选择６３４枚移植到４６只假孕受体．其中１４只妊娠产仔，妊娠率３０．４％．妊娠受体共移植胚胎２０７枚，产仔４３只，产仔率２０．８％．经ＰＣＲ法检测８只为ＢＬＧ阳性整合，整合率２５．８％．检测泌乳的转基因阳性雌性小鼠，其中一只经过用生物素标记的鼠抗人ｈＣＤ１４单克隆抗体与琼脂糖凝胶转移片段简易杂交检测。

A 898 bp XbaI KpnI fragment derived from the clone pBLG containing the sheep β lactoglobulin (BLG) 5′ flanking regions and signal sequences,and a 1245bp Eco RI XbaI fragment derived from phCD14 containing the human CD14 mature peptide encoding regions and 3′ noncoding regions both prepared by polymerase chain reaction (PCR),were fused within the EcoRI KpnI site of pUC19 to construct pBLG hCD14. The 2.1kb HindIII Eco RI fragment from pBLG hCD14 was microinjected into pronuclei of mouse eggs.The result showed that 84.2%(968/1149) of microinjected eggs survived.634 2 cell embryos developed from the microinjected eggs were transferred to 46 pseudopregnant recipients. 30.4%(14/46) of the recipients became pregnant and 43 mice were born. Of 31 survived mice, 25.8%(8/31) were indentified as positive mice for transgenes by PCR screening.Milk from nursing mice Day 9 after parturition were analyzed on 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and determinated by simplified Western blot with biotin mouse anti human CD14 monoclonal antibody following electrophoresed on 1% agros and transfromed to nitrocellulose (NC) member. The results showed that hCD14 was expressed in Tg002 transgenic mouse milk.