山羊β-酪蛋白启动子指导的通用表达载体及乳腺暂时表达系统  

Construction of a Base Expression Vector Directed by Goat β-casein Promoter and Its Transient Expression in Mammary Glands

作　　者：徐青[1] 扈荣良[1] 张守峰[1] 梁冠生[2] 

机构地区：[1]解放军军需大学军事兽医研究所,吉林长春130062 [2]解放军军需大学军事兽医系,吉林长春130062

出　　处：《中国兽医学报》2003年第6期576-580,共5页Chinese Journal of Veterinary Science

基　　金：吉林省科技发展计划资助项目 (2 0 0 10 10 6)

摘　　要：为了实现外源蛋白在动物乳腺中的特异性高效表达 ,利用山羊 β-酪蛋白 5′端 1.132 kb的侧翼调控序列、牛生长激素基因 poly A ( BGHPA)序列、基质结合区 ( matrix attachment region,MAR)和多克隆位点 ( MCS) ,构建了乳腺组织定位通用表达载体 p MRPA,鉴定正确后 ,将 2 .1kb的人组织型纤溶酶原激活剂 ( tissue- type plasm inogen activator,t PA) c DNA克隆到 p MRPA多克隆位点 ,获得重组表达载体 p MRPA- t PA。在不同的泌乳时间 ,使用不同的包裹剂 ,采用不同的剂量 ,将 p MRPA- t PA注入泌乳期山羊乳腺组织进行暂时表达。结果显示 ,所构建的乳腺组织特异性通用表达载体能够有效地指导目的基因的高效表达。通过乳腺暂时表达试验进一步发现 ,泌乳稳定期表达效果最好 ,t PA表达水平在 2 .0 2 0～ 6 .95 9mg/ L ;表达量随 DNA用量的增加而提高 ;使用普通包裹剂和裸质粒相比 ,普通包裹剂对表达效果无明显影响。本试验对乳腺暂时表达系统进行了系统性研究 。In order to express valuable proteins in animal mammary glands,a base vector containing goat β casein gene promoter,bovine growth hormone polyadenylation signal,matrix attachment region and multiple clone sites was constructed. After being fused with tissue plasminogen activator cDNA, the vector was injected directly into the lactating mammary glands of goats. The transient expression results indicated that the mammary specific expression vector driven by the goat β casein gene promoter could efficiently direct the expression of tPA in goat milk,ranging from 2 020 to 0 959 mg per l liter. It was further found that the tPA was secreted stably during later stage of lactation. The expression level had direct correlation with the amount of DNA injected. The common transfection reagents such as span glycerol did not effect on the gene expression in this study.

关 键 词：山羊β-酪蛋白启动子 表达载体 暂时表达系统 组织型纤溶酶原激活剂 乳腺生物反应器 药用蛋白 

分 类 号：TQ464.7[化学工程—制药化工] TQ460.38