表达人尿激酶原突变体的重组山羊乳腺特异性表达慢病毒载体的构建

Construction of Recombinant Lentivirus Expression Vector Expressing Human pro-Urokinase Mutant Specific for Mammary Gland of Goats

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、复制缺陷型人免疫缺陷病毒(HIV-1)的5'端长重复序列(LTR)、HIV-1ψ包装信号、HIV Rev反应元件、山羊β-酪蛋白调控序列、尿激酶原M13cDNA、ΔU3/3'LTR、牛生长激素(BGH)基因poly(A)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、中国仓鼠卵巢细胞及泌乳山羊乳腺注射证明其表达有效性。结果:酶切鉴定证实山羊乳腺特异性表达载体构建正确;将该载体转染细胞,采用溶圈法和Western印迹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:为在转基因动物乳腺中表达尿激酶原突变体奠定了基础。

Objective： To construct the recombinant lentivirus expression vector which can specifically express human pro-urokinase mutant in the mammary gland of goats and prove its specificity. Methods： Rous sarcoma wrus 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 β-casein genomic sequence and pro- UKM13 cDNA were used to construct lentivirus expression vector specific for mammary gland. 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. Results： The recombinant lentivirus expression vector was identified by restriction endonuclease, and the expression of pro-UKM13 was proved by fibrin plate assay and Western blot analysis. The transient expression results in- dicated that the mammary specific expression vector could efficiently direct the expression of pro-UKM13 in goat milk. Conclusion： The recombinant lentivirus expression vector can express in goat mammary gland, and this study provides the basis for establishing transgenic animal which could specific express pro-UKM13 in mammary by recombinant lentivirus.