目的：应用基因工程技术构建含大鼠β-防御素2(rat beta defensin 2，rBD-2)目的基因的真核重组质粒，通过脂质体法转染大鼠角膜上皮细胞，检测目的基因在转染细胞中的表达，探讨应用该载体获得重组rBD-2在眼表细胞中表达的可行性，并为进一步研究rBD-2的体内外抗微生物活性提供实验基础，以期为感染性角膜病防治提供新方法。

方法：将采用PAS(PCR-based Accurate Synthesis)的方法合成rBD-2 DNA片段连接到真核表达载体pIRES2-ZsGreen1的XhoⅠ与BamHⅠ酶切位点之间，构建pIRES2-ZsGreen1-rBD-2真核重组表达载体，重组质粒转化大肠杆菌DH5a感受态细胞，卡那霉素筛选出阳性克隆子，经酶切、测序鉴定重组载体构建成功后，采用脂质体法转染大鼠角膜上皮细胞，此处实验分为三组即重组载体pIRES2-ZsGreen1-rBD-2转染的大鼠角膜上皮细胞组、未转染的空细胞组以及空载体pIRES2-ZsGreen1所转染的大鼠角膜上皮细胞组，在倒置荧光显微镜下观察细胞转染情况，最后经实时荧光定量RT-PCR相对定量法检测各组转染细胞中rBD-2基因mRNA的表达差异。

结果：成功构建pIRES2-ZsGreen1-rBD-2真核重组质粒，应用实时荧光定量RT-PCR相对定量法检测到重组质粒pIRES2-ZsGreen1-rBD-2转染组的大鼠角膜上皮细胞中rBD-2基因的mRNA表达水平明显多于另两组。

结论：应用基因工程技术构建的rBD-2真核表达载体pIRES2-ZsGreen1-rBD-2，通过脂质体法转染大鼠角膜上皮细胞，能够使外源rBD-2基因在大鼠角膜上皮细胞中被转录成mRNA。

AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 in vitro and in vivo and to assess the probability of defensins as a new application for infectious corneal diseases in the future.

METHODS: The synthetic rBD-2 DNA fragment was inserted between the XhoI and BamHI restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into E.coli DH5α, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR.

RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR.

CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

湖北省卫生和计划生育科研项目(No.WJ2015MB194)