中国兽医科学2011，Vol.41Issue(8)：806-810,5.
奶牛乳腺特异性表达人溶菌酶基因载体的构建与表达
Construction and expression of bovine mammary gland specific expressing vectors with human lysozyme gene
摘要
Abstract
To explore the feasibility of decreasing mastitis in cows using transgenic animal techniques, according to the mRNA sequence of human lysozyme（hLZ）（NM_000239.2）, using RT-PCR technique, a 406 bp hLZ fragment was amplified from total RNA of human placenta tissues. Using bovine beta-casein regulatory elements as promoter,two mammary gland-specific expressing hLZ gene vectors pBCN5.2-hLZ 1. 1pA-EGFP-neo（13.6 kb） and pBCN-3.8-hLZ-1, lpA-EGFP-neo（12.2 kb） were constructed,after several times of digesting-ligasing-transfecting, and identification. The vectors were transfected into Bcap cells with lipofectamine respectively,and green fluorescence protein was observed at hour 24 post-transfection. Total RNA of these transfected cells was purified and examined by qRT-PCR, and the relative expression were 0.89±0.04 and 0.62±0.05 respectively. Then lined pBCNS. 2-hLZ1.1pA EGFP-neo DNA was transfected into Bcap cells and selected with G418. Total proteins were isolated from positive cells and assayed by SDS-PAGE,a 14 ku new protein was observed comparing with control cells, Western-blot analysis revealed a 14 ku specific band. This work is of great importance for producing cows naturally against mastitis transgenic in the near future.关键词
奶牛/乳腺特异性表达/人溶菌酶基因/转基因载体Key words
bovine/mammary gland-specific expression/human lysozyme gene/transgenic vector分类
农业科技引用本文复制引用
刘庆友,刘晟,王丹,李辉,李湘萍,罗婵,陆凤花,石德顺..奶牛乳腺特异性表达人溶菌酶基因载体的构建与表达[J].中国兽医科学,2011,41(8):806-810,5.基金项目
国家高技术研究发展计划（863)项目（2007AAL00505) （）
国家转基因重大专项（2008ZX08007003) （）
国家自然科学基金项目（30960251) （）
广西自然科学基金项目（0991007Z) （）
