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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
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)
关键词
抗菌肽
重组逆转录病毒
转染
包装
表达
antimicrobial peptide
recombinant retrovirus
transfection
package
expression
