山羊乳腺特异性表达慢病毒载体的构建与验证

[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-1ψ卫包装信号、HIVRev反应元件、山羊B-casein调控序列、pro—UKM13、△U3／3’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Westernblot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。

Construction and Verification of Lentivirus Expression Vector Specific for Mammary Gland

[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.