抗菌肽重组逆转录病毒载体的构建、重组病毒的包装及其在牛奶中的稳定表达  

Construction of recombinant retroviral vector of antimicrobial peptide genes and expression in mammary gland

作　　者：张锦霞[1] 张守峰[1] 郭学军[1] 扈荣良[1] 

机构地区：[1]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2008年第12期1433-1437,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30471280)

摘　　要：将含有双抗菌肽基因的表达盒插入到逆转录病毒载体pLN—bcp—LK（R）中，获得了含有双抗菌肽基因的重组逆转录病毒载体pLN—bcp-LfcinB-CE-MA（R），利用脂质体转染PA317包装细胞，G418进行筛选得到了阳性细胞克隆，克隆扩大培养后，用病毒上清感染NIH3T3细胞进行滴度测定，最高滴度为1×10^4CFU／mL，用病毒上清转染妊娠中后期奶牛乳腺组织，产犊后采集的奶样经琼脂板溶圈法和Tricine-SDS-PAGE检测，结果表明乳汁中表达的抗菌肽对大肠杆菌和金黄色葡萄球菌均具有明显地抑菌活性，并能实现相对稳定的表达，表达可持续30-45d。Antimicrobial peptide genes were cloned into the retroviral vector pLN-bcp-LK（R） to construct the recombinant retroviral vector pLN-bcp-LfcinB-CE-MA（R）. The vector were transfected into PA317 cells for packaging by liposomal transfection,after G418 selection for 2 weeks,G418-resistant PA317 colonies were obtained and amplified. The supernatant of cell culture was harvested and infected NIH3T3 cells,as were to measure the viral titer of recombinant retrovirus, results showed that the highest titer of viral supernatant was 1 × 10^4 CFU/mL. The result of Tricine-SDS-PAGE and inhibition zone assay showed that antimicrobial peptides genes had been expressed continuously in milk of the post partum cow,the expression of antibacterial proteins persisted for 30-45 days. Inhibition zone assay suggested that the milk samples also revealed the bacteriostatic activity.

关 键 词：抗菌肽 重组逆转录病毒 转染 包装 表达 

分 类 号：Q78[生物学—分子生物学] S823[农业科学—畜牧学]