慢病毒介导抗菌肽PR-39基因在兔骨髓间充质干细胞中的表达及抗菌活性检测

The expression and antibacterial activity of antimicrobial peptides PR-39 in rabbit bone mesenchymal stem cells mediated by lentiviral vector

目的构建脯氨酸精氨酸抗菌肽（proline-arginine rich 39-amino acid peptide,PR-39）慢病毒载体,并转染兔骨髓间充质干细胞（bone mesenchymal stem cells,BMSC）,检测其在BMSC中的表达及抗菌活性。方法PCR扩增目的克隆,并连接入慢病毒载体质粒pGC-FU中,与包装质粒pHelper1.0、pHelper2.0在脂质体2000介导下共转染293T细胞生产慢病毒颗粒,Real-TimePCR法检测其滴度;分离培养BMSC,BMSC转染PR-39慢病毒,荧光显微镜观察及流式细胞仪检测其转染效率,RT-PCR检测PR-39基因表达,并检测PR-39抗菌活性。结果成功包装出滴度为2×10~9 TU/mL的PR-39慢病毒载体,并转染BMSC,转染72 h后可观察到细胞呈现绿色荧光,转染效率为74.09%,同时RT-PCR检测BMSC表达PR-39基因,其上清液对金黄色葡萄球菌有明显的抑制作用,抑菌率为98.43%（P〈0.05）。结论在慢病毒介导下,BMSC成功表达分泌具有较强抗菌活性的抗菌肽PR-39。

Objective To construct the proline-arginine rich 39-amino acid peptide（PR-39） lentiviral vector, and introduce it into bone mesenchymal stem cells（BMSC）, the expression and antibacterial activity of PR-39 was detected. Methods The CDS sequence of PR-39 was amplified by PCR from pBluescript II SK-PR-39, and then the gene was recombined into the transfer plasmid pGC-FU. The three plasmids of lentiviral vector, transfer plasmid pGC-FU-PR-39, packaging plasmid pHelperl.0, pHelper2.0, which were cotransfected to 293T cells mediated by lipofectamin 2000 to produce lentiviral particles. The tite of viral particles was detected by real-time PCR. The rabbit BMSC were prepared, and then were infected by PR-39 lentiviral particles. The transfection efficiency was observed with fluorescent microscope and flow cytometry, and the expression of PR-39 was detected by RT-PCR, and the antibacterial activity also was investigated. Results The tite of viral particles was 2× 109 TU/mL, and it was showed transfection efficiency was 74.09%. PR-39 gene was expressed successfully in BMSC, and PR-39 inhibited the staphylococcus aureus significantly, and bacteriostatic rate was 98.43%（P〈0.05）. Conclusion Antimicrobial peptides PR-39 is expressed successfully in rabbit BMSC mediated by lentiviral vector, and it shows significant antibacterial activity.