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为探讨应用转基因动物技术降低奶牛乳腺炎发病的可行性,参照人溶菌酶(human lysozyme,hLZ)基因序列(NM_000239.2)设计引物,采用RT-PCR方法从人胎盘组织中成功扩增克隆了406bp的hLZ基因成熟肽片段。应用娟姗牛酪蛋白乳腺特异表达调控元件,经过多步酶切、连接、转化、筛选、鉴定,构建成奶牛乳腺表达hLZ的载体pBCN5.2-hLZ-1.1pA-EGFP-neo(13.6kb)和pBCN-3.8-hLZ-1.1pA-EGFP-neo(12.2kb)。将构建的2个载体瞬时转染Bcap细胞,24h后可观察到EGFP的表达;通过实时定量PCR检测比较2个载体hLZ的表达丰度,结果,相对表达量分别为0.89±0.04和0.62±0.05。将线性化的pB-CN5.2-hLZ-1.1pA-EGFP-neo载体转染Bcap细胞,筛选后获得阳性细胞克隆,纯化阳性细胞总蛋白进行SDS-PAGE分析,与空白对照相比,检测到14ku的新蛋白条带。Western-blot分析中亦出现14ku的特异性条带。证实构建的载体能够正常表达hLZ基因,这为进一步研究获得自然抵抗乳腺炎的转基因奶牛奠定了工作基础。
To explore the feasibility of using transgenic animal technology to reduce the incidence of dairy cow mastitis, primers were designed according to the human lysozyme (hLZ) gene sequence (NM_000239.2) and the clones were successfully amplified from human placenta by RT-PCR 406 bp hLZ gene mature peptide fragment. The vector pBCN5.2-hLZ-1.1pA-EGFP-neo (13.6kb) was constructed by using milk-cow mammary gland mammary gland-specific expression regulatory elements. After several steps of digestion, ligation and transformation, screening and identification, pBCN-3.8-hLZ-1.1 pA-EGFP-neo (12.2 kb). The constructed two vectors were transiently transfected into Bcap cells, and the expression of EGFP was observed after 24 hours. The expression abundance of hLZ in two vectors was compared by real-time quantitative PCR, and the relative expression levels were 0.89 ± 0.04 and 0.62 ± 0.05 . The linearized pB-CN5.2-hLZ-1.1pA-EGFP-neo vector was transfected into Bcap cells. After screening, positive clones were obtained and the total proteins were purified by SDS-PAGE. Compared with the blank control, New protein band of 14ku. Western-blot analysis also appeared 14ku specific bands. It was confirmed that the constructed vector can express hLZ gene normally, which lays a foundation for further research on transgenic cows that naturally resist mastitis.