β-防御素2基因腺病毒表达载体的构建与真核表达

目的：探讨重组腺病毒载体在真核细胞中稳定表达阳离子抗菌肽——β-防御素2的可行性。方法：采用RT-PCR方法扩增得到大鼠β-防御素2（rBD2）基因片段，定向插入到腺病毒穿梭质粒pShuttle-CMV中，重组质粒pShuttle-CMV-rBD2转化E．coli BJ5183-AD-1后，与腺病毒基因组质粒pAdEasy-1发生同源重组，重组质粒pAdEasy-rBD2转染293细胞进行腺病毒表达载体的包装。将包装成功的腺病毒感染cos-7细胞，并建立SD大鼠腺病毒感染模型，进行β-防御素2多肽的体外和体内表达，采用Western blot与免疫组化方法检测其表达。结果：重组腺病毒表达载体构建成功，包含大鼠β-防御素2基因，滴度达到10^9PFU／ml，Western blot与免疫组化方法分别检测到β-防御素2在SD大鼠体外、体内的表达。结论：重组腺病毒载体能在真核细胞中稳定表达阳离子抗菌肽——β-防御素2。

Construction and expression of recombinant adenovirus expression vector of β-defensin-2

OBJECTIVE: To explore the possibility of stable expression of cationic peptide beta-defensin-2 in eukaryocytes with adenovirus vector. METHODS: The rat beta-defensin-2 (rBD2) gene was cloned at the downstream of CMV promoter of the adenoviral shuttle plasmid pShuttle-CMV. Then pShuttle-CMV-rBD2 was transformed into E. coli BJ5183-AD-1, in which recombination occurred between plasmids and pAdEasy-1 to construct pAdEasy-rBD2. After confirmation by endonuclease, linear pAdEasy-rBD2 was transformed into 292 cells to obtain packaged adenoviral expression vector, which was used to infect cos-7 cells and to establish respiratory adenovirus infection model of rat. The in vivo and in vitro expression activity of recombinant adenovirus was detected by Western blot and immunohistochemistry. RESULT: The inserted DNA of pShuttle-CMV-rBD2 consisted of rat beta-defensin-2 gene. The pathological effect of infected cells, electronic microscopic observation and PCR showed that the recombinant adenovirus vector was constructed successfully. The concentration of the adenovirus was 10(9) PFU/ml. The vector expressed rat beta-defensin-2 efficiently in vivo and in vitro. CONCLUSION: The recombinant adenovirus vector can express cationic peptide beta-defensin-2 in eukaryocytes.