摘要
目的构建人β防御素2（hBD-2）的真核表达载体PLXSN的重组质粒pLXSN-hBD2，将其转染至大鼠子宫内膜异位症（EMS）模型的局部异位病灶组织，并检测hBD-2基因和蛋白表达。方法利用双酶切定位定向克隆技术，将hBD-2基因片段连接于pLXSN质粒的EcoRI和BamHI两个酶切位点之间，构建重组质粒pLXSN-hBD2。自体内膜移植法建立大鼠EMS模型，将pLXSN-hBD2局部多点注射至其局部异位病灶组织，Western印迹检测局部病灶组织中的hBD-2蛋白表达。结果经过重组质粒的鉴定电泳以及测序鉴定，设计合成的基因片段序列与GenBank中hBD-2cDNA序列完全相同，其中碱基54-248为目的基因hBD-2序列。Western印迹证实大鼠EMS模型异位病灶局部有hBD．2蛋白的表达。结论通过局部多点注射的方法，可将携带hBD-2基因的重组质粒pLXSN-hBD2成功转染至大鼠EMS异位病灶组织中，并获得hBD-2蛋白的表达。
Objective To construct pLXSN-hBD2, the human defensin-2 （hBD-2） eukaryotic pLXSN recombinant plasmid, thus allowing for transfection to the ectopic loci of endometriosis in rats （EMS） and subsequent assessment of hBD-2 gene and protein expression. Methods The hBD-2 gene fragment was linked to the digestion sites of EcoR I and BamH I of the pLXSN plasmid for construction of the recombinant plasmid pLXSN-hBD2 by using double enzyme positioning and directional cloning technology. The EMS rat model was constructed by autologous membrane transplantation. This was followed by multi-foci injection of pLXSN-hBD2 to the ectopic foci, thus allowing for determination of hBD2 protein expression in ectopic endometrium by Western blotting. Results Following recombinant plasmid electrophoresis and sequencing, the designed gene fragment possessed an identical sequence with that of hBD - 2 cDNA in GenBank, in which the 54-248 base pairs were consistent with the sequence of hBD- 2 target gene. The expression of hBD-2 protein was observed in ectopic foci, as evidenced by Western blotting. Conclusion By multi-foci injection, the recombinant plasmid pLXSN-hBD2 containing hBD-2 gene can be successfully transfected to the endometriosis tissues in rats for harvesting hBD-2 protein.
出处
《中华生物医学工程杂志》
CAS
2013年第3期204-209,共6页
Chinese Journal of Biomedical Engineering
基金
国家自然科学基金面上项目（81070472）
关键词
防御素2
人
重组质粒
Defensin-2
Human
Recombinant plasmid
