过表达驴小肽转运载体1 CHO细胞的构建  

Construction of Chinese hamster ovary (CHO) cells over-expressing donkey oligopeptide transporter 1

作　　者：周苗苗 曹贵玲[1] 刘桂芹[1] 刘文强[1] 朱明霞[1] 王长法 ZHOU Miaomiao;CAO Guiling;LIU Guiqin;LIU Wenqiang;ZHU Mingxia;WANG Changfa(College of Agronomy/Institute of Donkey High-Efficiency Breeding and Ecological Feeding,Liaocheng University,Liaocheng 252000,China)

机构地区：[1]聊城大学农学院/毛驴高效繁育与生态饲养研究院,山东聊城252000

出　　处：《黑龙江畜牧兽医》2021年第19期127-130,共4页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31902177);山东省“泰山产业领军人才”项目(LJNY201713);聊城大学博士科研启动基金项目(318051816)。

摘　　要：为了研究驴小肽转运载体1(oligopeptide transporter1,PepT1)的转运功能,试验取驴肝脏组织,对PepT1基因进行克隆构建了驴PepT1的表达载体pcDNA3.1-PepT1,并将其转染至中华仓鼠卵巢(CHO)细胞中,采用Western-blot方法检测转染效率,然后以未转染的CHO细胞作对照进一步检测转染后CHO细胞对β-丙氨酸-赖氨酸-N-7-氨基-4-甲基香豆素-3-乙酸(β-Ala-Lys-AMCA)的摄取情况。结果表明:成功扩增出驴PepT1基因CDS序列,并构建了表达载体pcDNA3.1-PepT1;转染pcDNA3.1-PepT124 h后,在CHO细胞中检测到过表达的驴PepT1蛋白,且过表达驴PepT1蛋白的CHO细胞对β-Ala-Lys-AMCA的摄取量显著高于对照组(P<0.05)。说明成功构建了pcDNA3.1-PepT1表达载体并转染至CHO细胞中,实现了驴PepT1蛋白的瞬时过表达。In order to study the transport function of donkey oligopeptide transporter 1(PepT1),in the experiment, cloning of PepT1 gene from donkey liver tissue, the expression vector pcDNA3.1-PepT1 of donkey PepT1 was constructed and transfected into Chinese hamster ovary cells(CHO);transfection efficiency was detected by Western-blot;then untransfected CHO cells was used as a control to further detect the uptake of β-alanine-lysine-N-7-amino-4-methylcoumarin-3-acetic acid(β-Ala-Lys-AMCA) by the transfected CHO cells. The results showed that CDS sequence of donkey PepT1 gene was amplified successfully, and the pcDNA3.1-PepT1 expression vector was constructed;24 h post the transfection of pcDNA3.1-PepT1,the over-expressing PepT1 protein was detected in the transfected CHO cells, and the uptake of β-Ala-Lys-AMCA was significantly increased in the CHO cells over-expressing PepT1 than that in control group(P<0.05). The results suggested that pcDNA3.1-PepT1 expression vector was successfully constructed and transfected into CHO cells, which achieved the transient over-expression of donkey PepT1 protein.

关 键 词：驴 小肽转运载体1 表达载体 中华仓鼠卵巢细胞 转运 

分 类 号：S822[农业科学—畜牧学] Q813.5[农业科学—畜牧兽医]