β-防御素2基因腺病毒表达载体的构建与真核表达

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

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(rBD 2)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-rBD 2转化E.coli B J5183-AD-1后,与腺病毒基因组质粒pA dE asy-1发生同源重组,重组质粒pA dE asy-rBD 2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W estern b lot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109PFU/m l,W estern b lot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。

Objective： To explore the possibility of stable expression of cationic peptide β-defensin-2 in eukaryocytes with adenovirus vector. Methods： The rat β-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. Results： The inserted DNA of pShuttle-CMV-rBD2 consisted of rat β-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 10^9PFU/ml. The vector expressed rat β-defensin-2 efficiently in vivo and in vitro. Conclusion： The recombinant adenovirus vector can express cationic peptide β-defensin-2 in eukaryocytes.