携抗菌肽PR39基因重组腺病毒的构建及抗胞内伤寒菌研究

目的构建抗菌肽PR39基因腺病毒表达载体,观察其在小鼠巨噬细胞RAW264.7中的表达及抗菌活性。方法PCR扩增抗菌肽PR39成熟肽基因,插入腺病毒穿梭载体pAdTrack-CMV中,经PmeI线性化后与腺病毒骨架(pAdEasy-1)共转化大肠杆菌BJ5183,通过细菌内同源重组产生重组腺病毒载体(pAd-PR39)。重组腺病毒载体经PacI线性化后转染293细胞,包装出重组腺病毒(Ad-PR39)。利用重组腺病毒感染体外培养的小鼠巨噬细胞RAW264.7,采用RT-PCR和免疫细胞化学检测目的基因的表达,并对其抗菌活性进行初步检测。结果先后构建了携抗菌肽PR39基因的穿梭载体和重组病毒载体,包装出重组腺病毒,并成功感染小鼠巨噬细胞RAW264.7。感染重组腺病毒的小鼠巨噬细胞RAW264.7能有效表达抗菌肽PR39。与对照(10.8±2.1CFU/cell)相比,感染重组腺病毒的小鼠巨噬细胞RAW264.7具有更强的杀灭胞内伤寒菌的能力(7.2±1.8CFU/cell)。结论构建的重组腺病毒能够感染小鼠巨噬细胞RAW264.7并发挥抗菌作用,为抗菌肽PR39在胞内菌感染基因治疗中的应用奠定了实验基础。

Construction of recombinant adenovirus expressing antimicrobial peptide PR39 and its antimicrobial activity

To construct an adenovirus expression vector encoding antimicrobial peptide PR39, and to observe its expres- sion and bioactivity in mouse macrophage RAW264.7 cells, PR39 gene was amplified with PCR and inserted into adenovirus shuttle vector, which was co-transformed into E. coli BJ5183 with pAdEasy-1 after linearization by PmeI. In E. coli BJ5183, recombination occurred to obtain recombinant adenovirus vector pAd PR39, which was used to transfect 293 cells after linear ized by PacI to obtain recombinant adenovirus Ad-PR39. RT-PCR and immunocytochemistry was used to detect the expression of PR39 gene in mouse macrophage RAW264.7. Recombinant adenovirus encoding antimicrobial peptide PR39 was successfully constructed which could infect RAW264, 7 cell efficiently by RT-PCR and immunocytochemistry detection of PR39 expression. RAW 264.7 cells infected with recombinant adenovirus Ad-PR39 showed strong antibacterial ability. These results demon- strates that recombinant adenovirus Ad-PR39 can express antimicrobial peptide PR39 in mouse macrophage RAW264.7 cell and enhance its antimicrobial ability, which established a foundation for gene therapy of intracellular bacterial infection with antimi- erobial peptides.