HBD3基因真核表达质粒载体的构建及其体外表达和活性鉴定

Construction and in expression of eukaryotic expression plasmid containing HBD3 gene

目的构建人β防御素3(HBD3)基因真核表达质粒载体,观察其在大鼠骨髓间充质干细胞(BMSCs)中的表达,并检测其活性。方法利用RT-PCR方法从牙龈组织中扩增出HBD3基因片段,通过DNA重组技术将基因片段重组于pVIVO1-mcs真核表达质粒载体上,构建成pVIVO1-HBD3重组质粒载体,分别用酶切电泳分析及DNA测序的方法对重组质粒载体进行鉴定。运用jetPEI转染试剂将重组质粒转染BMSCs后,RT-PCR、免疫荧光和蛋白质印迹法检测目的基因的表达。取转染重组质粒的BMSCs无血清上清的浓缩液,采用K-B纸片扩散法进行抗菌活性实验。结果 (1)酶切电泳分析得到相应的目的片段,大小与理论计算值一致,DNA测序证实目的基因序列正确无突变,成功构建真核表达质粒载体pVIVO1-HBD3。(2)RT-PCR和免疫荧光、蛋白质印迹法分别从mRNA和蛋白质2个水平证实转染的BMSCs高表达HBD3。(3)转染重组质粒的BMSCs无血清上清的浓缩液对白色念珠菌、大肠埃希菌、绿脓杆菌、金黄色葡萄球菌和枯草芽孢杆菌产生明显的抑菌环,证实了表达分泌出的HBD3的抗菌活性。结论构建了pVIVO1-HBD3基因真核表达质粒载体,证实其转染BMSCs后基因的表达和活性。

Objective To further determine the possible effect on antimicrobial activity,plasmid vector containing recombinant human beta defensins 3（HBD3）gene was constructed and the expression of exogenous gene in transformed bone marrow-derived mesenchymal stem cells（BMSCs）was observed.Methods By using RT-PCR obtained HBD3 from gingiva tissue,then recombinated the gene to plasmid pVIVO1-mcs.The recombinated plasmid vector was named as pVIVO1-HBD3 and identified by restriction enzyme analysis and DNA sequencin.After the recombinated plasmid transformed BMSCs,expression of HBD3 in BMSCs was detected by RT-PCR,immunofluorescence and Western blot,and the function was determined by Kleihauer-Betke（K-B） test.Results We successfully constructed recombinant plasmid vector that expressed HBD3.The expression of HBD3 were confirmed by RT-PCR,immunofluorescence and Western blot on transformed BMSCs.The function of HBD3 were confirmed by K-B test.Conclusion The established BMSCs that overexpressed HBD3 provide a new strategy of gene therapy to promote wound healing,especially the infected one.