【摘要】 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。更多还原

【Abstract】 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 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 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 indicated 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.更多还原