[Objective] The research aimed to construct the recombinant lentivirus expression vector that can specifically express pro-UKm in the mammary gland and prove its specificity. [ Method ] Rous Sarcoma Virus enhancer/promoter, modified HIV-1 5' and 3' Long Terminal Repeat (LTR) ,HIV-1 psi(Ψ) packageing sequence,HIV Rev response element were amplified by PCR,then partial goat β-casein promoter,partial goat P-casein genomic sequence and pro-UK_(M13) cDNA were used to construct lentivirus expression vector specific for mammary gland. And its expression specificity was proved by transfection the MCF-7 and CHO cell in vitro and directly injecting the vector into the lactating mammary glands of goats. [Result] The recombinant expression lentivirus expression vector was identified by restriction endonuclease,and the expression of pro-UK_(M13), was proved by fibrin plate assay and western blot analysis. The transient expression results indicated that the mammary specific expression vector could efficiently direct the expression of pro-UK_(M13) in goat milk. The recombinant lentivirus expression vector can express in milk and goat mammary gland. [Conclusion] This study provids the basis for establishing transgenic animal that can express pro-UK_(M13) in mammary by recombinant lentivirus.%[目的]构建山羊乳腺特异性表达尿激酶原突变体的重组慢病毒载体,证明其表达的有效性.[方法]将劳氏肉瘤病毒增强子/启动子、复制缺陷型艾滋病病毒(HIV-1)的5'端长重复序列(LTR)、HIV-1 Ψ包装信号、HIV Rev 反应元件、山羊β-casein调控序列、pro-UK_(M13)、ΔU3/3'LTR依次连接,构建乳腺特异性表达的慢病毒载体;通过体外转染人乳腺癌细胞系(MCF-7)、仓鼠卵巢细胞(CHO)和泌乳山羊乳腺注射证明其表达有效性.[结果]构建的山羊乳腺特异性表达载体酶切鉴定是正确的;利用该载体转染细胞,采用溶圈法和Western blot检测证实了其表达的有效性;慢病毒载体注射到泌乳山羊的乳腺,在乳汁中也检测到了尿激酶原的表达.[结论]为在转基因动物的乳腺中表达尿激酶原突变体奠定了基础.
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