奶牛气管抗菌肽(TAP)基因乳腺特异性表达载体的构建与鉴定

Construction and identification of mammary gland specific expressing vector of bovine tracheal antimicrobial peptide(TAP)

【目的】构建奶牛气管抗菌肽(TAP)基因乳腺特异性表达载体。【方法】根据GenBank中牛TAP基因cDNA序列(GenBank号:NM_174776)设计引物,从奶牛乳腺组织中RT-PCR扩增TAP基因编码序列,将其克隆到pMD19-T(simple)载体中测序。重组质粒pMD-TAP经EcoRⅠ+KpnⅠ双酶切回收目的基因片段,亚克隆到携带增强型绿色荧光蛋白的通用型乳腺特异性表达载体pBLG-EGFP中,采用脂质体法转染奶牛乳腺上皮细胞(bMEC)、COS-7细胞,并注射泌乳家兔乳腺组织,荧光显微镜检测转染细胞中绿色荧光蛋白的表达,半定量RT-PCR检测家兔乳腺组织中TAP mRNA的表达。【结果】成功构建了乳腺特异性表达载体pBLG-EGFP-TAP,该载体可在bMEC中特异性地表达绿色荧光蛋白;半定量RT-PCR检测发现,转染pBLG-EGFP-TAP可显著提高家兔乳腺组织中TAPmRNA的表达水平。【结论】成功构建了乳腺特异性表达载体pBLG-EGFP-TAP,为进一步研究奶牛TAP基因的表达特性及利用基因工程技术防治奶牛乳房炎提供了重要材料。

【Objective】 This study was to construct the mammary gland specific expressing vector of bovine trachea antimicrobial peptide（TAP） gene.【Method】 TAP gene from the mammary gland tissue of dairy cattle was amplified by RT-PCR using a pair of primers according to the sequence in GenBank（NM_174776）,and then cloned into the pMD19-T simple vector.The fragment TAP was extracted by digesting the recombinant plasmid with EcoR Ⅰ＋Kpn Ⅰ before being inserted into the pBLG-EGFP vector to construct mammary gland specific expressing vector,pBLG-EGFP-TAP.Finally,the constructed plasmid pBLG-EGFP-TAP was transfected into bovine mammary epithelial cells（bMEC）,COS-7 cells and rabbit mammary gland tissue by lipofectin transfection,then green fluorescent protein（EGFP） expression in cells was detected under fluorescence microscopy,and TAP mRNA expression in tissue was determined by semi-quantity RT-PCR.【Result】 The results showed that EGFP was specially expressed in transfected bMECs,in same time,TAP mRNA expression level was significantly increased in transfected mammary gland tissue of rabbit.【Conclusion】 The results indicated that the mammary gland specific expressing vector pBLG-EGFP-TAP was successfully constructed,providing important material for further investigation of expression feature of TAP and prevention mastitis by gene therapy.