人α-乳清蛋白基因的克隆及其在转基因小鼠中高效表达

从粘粒文库中筛选出人α-乳清蛋白基因,构建9.5 kb的转基因表达载体.利用显微注射的方法获得68只F₀代小鼠,经PCR检测和DNA印迹分析证实有8只小鼠（4♂,4♀）为整合人α-乳清蛋白基因的转基因阳性小鼠,整合率为11.7%,整合拷贝数在1至8之间.利用SDS-聚丙烯酰胺凝胶电泳检测和蛋白质印迹分析,4只雌性F₀代转基因阳性小鼠全部表达了人α-乳清蛋白.放射免疫测定法测定,含量分别为0.62 g/L、0.48 g/L、0.56 g/L、3.21 g/L;同时测定F₀代50号转基因公鼠的后代阳性母鼠（50-2号）乳样中人α-乳清蛋白含量也达到1.03 g/L,证明由原代转基因公鼠遗传给后代的人α-乳清蛋白基因亦获得了稳定的表达.所构建的人α-乳清蛋白转基因载体具有结构较小,表达量高,可以稳定遗传等优点.为利用人α-乳清蛋白基因改善牛乳成分和品质奠定了基础.     

鸡形目鸟类成熟胸肌中特异性表达的fTnT同工异构型及其生理学意义

脊椎动物中的肌钙蛋白T（tropnin T,TnT）分为心肌型TnT（cardiac TnT,cTnT）、快肌型TnT（fast skeletal TnT,fTnT）和慢肌型TnT（slow skeletal TnT,sTnT）,且每种TnT又因mRNA可变剪接(alternative mRNA splicing)形成了多种同工异构型,其中fTnT的同工异构型形式最为复杂。某些鸟类如鸡形目鸟类的成熟快肌（尤其是胸部快肌）中特异性表达的TnT同工异构型有如下特点：（1）N端区含有过渡金属离子结合位点——Tx元件（一般为4~7个重复的H-E/A-E-A-H序列）;（2）与哺乳动物及雏鸟fTnT相比,其C端区外显子16有很高的表达率。本文还就鸡形目鸟类成熟胸肌中表达的fTnT同工异构型可能具有的生理学意义及应用前景进行了探讨。 The Fast TnT Isoforms Specifically Expressed in Avian Adult Pectoral Muscles of Galliforms and Physiological Significance DUAN Ying-li,YU Shu-yang,LI Ning National Laboratories for Agrobiotechnology,China Agricultural University,Beijing 100094,China Abstract:Three homologous genes have evolved to encode the cardiac,slow and fast skeletal muscle troponin Ts(TnTs) in the vertebrate.Multiple isoforms in each type of TnT are generated through alternative mRNA splicing during the development and the modality of the fast skeletal TnT isoforms is the most complex.The TnT isoforms specifically expressed in avian adult fast skeletal muscle (especially in the adult pectoral muscle) of Galliforms have been characterized as follows:1.There exist a cluster of transition metal ion binding sites ［generally 4~7 repeats of a sequence motif His-(Glu/Ala)- Glu-Ala-His,designated as Tx］ in the NH2-terminal variable region.2.Compared with mammalian TnT and the neonatal or young avian TnT,these avian pectoral muscle TnTs prefer to express exon 16 in the COOH-terminal variable region.Furthermore,possible effects of the pectoral fTnT isoforms on the physiological activity are discussed in this article. Key words：Aves; troponin T; isoform