猪β防御素-1基因乳腺特异性表达载体的构建

Construction of marker removable,mammary specific porcine β-defensin 1 expression vector

为构建标记基因可去除的β防御素-1(pBD-1)基因乳腺特异性表达载体,利用RT-PCR方法从猪脾脏中克隆获得pBD-1基因,再插入乳腺特异性表达骨架载体pBC1-neo中;经PCR、酶切电泳以及测序鉴定,确认成功构建出乳腺特异性表达pBD-1的载体。进一步将上述功能片段亚克隆至能将标记基因全部去除的骨架载体MCS-3s-loxP-GFP中,成功构建了一种筛选标记可全部去除的乳腺特异性3s-loxP-GFP-pBC1-pBD1转基因载体。该载体为研究pBD-1蛋白的抗菌活性、抗菌机制,以及创制乳腺高效表达pBD-1的转基因猪育种新材料和通过哺乳以提高新生仔猪抗病力奠定了基础。

Porcine β-defensin 1 （pBD-1） is a cationic peptide, widely spread in porcine organs, and plays an important role in porcine disease defence. In order to construct maker removable and mammary gland specific pBD-l expression vec- tor, the present study firstly performed RT-PCR to obtain coding sequence of pBD-1 gene from porcine spleen, and sub- sequently inserted the pBD-1 fragment into pBCl-neo. PCR, enzymatic digestion and seqencing results confirmed that the mammary gland specific pBD-1 vector was successfully constructed. Then, the combined fragment （including 5~ and 3＇ regulating region of β-casein and pBD-1） was moved into MCS-3s-loxP-GFP, and the marker removable and mammary gland specific expression vector 3s-loxP-GFP-pBCI-pBD1 was constructed. These results may pave the way to study anti- microbia activities and its mechanism of the pBD-1, and also are helpful to make marker-free transgenic porcine mammary bioreactors producing pBD-1, which would improve the immuno function of defensing against pathogenic bacteria, thereby increase the new born natural disease resistance.