CAJ | 학술논문

目的构建大鼠中介素(IMD)基因真核表达载体IMD-pCDNA3.1(+),以超声微泡法介导其在大鼠体内肾脏局部高表达.方法用HindⅢ和EcoRI双酶切IMD基因和目的载体pCDNA3.1(+),连接制备真核表达载体IMD-pCDNA3.1(+),命名为IMD-pCDNA3.1(+),并对重组表达载体进行酶切、测序鉴定.18只雄性Wistar大鼠随机分为未转染组、空质粒转染组以及IMD基因转染组,每组6只,采用超声微泡介导技术转染大鼠肾脏.RT-PCR、Western blotting检测IMD表达.结果经限制性酶切鉴定及测序分析证实IMD-pCDNA3.1(+)载体序列正确;3组大鼠肝、肾功能差异均无统计学意义(P均＞0.05);实验各组均未见明显肾小球及肾小管、间质损害;半定量RT-PCR、Western blotting检测显示:IMD转基因组肾组织IMD mRNA及其蛋白表达较空质粒转染组和未转染组明显上调.结论 IMD-pCDNA3.1(+)表达载体构建成功,并在大鼠肾脏内成功表达.
목적 구건대서중개소(IMD)기인진핵표체재체IMD-pCDNA3.1(+),이초성미포법개도기재대서체내신장국부고표체.방법 용HindⅢ화EcoRI쌍매절IMD기인화목적재체pCDNA3.1(+),련접제비진핵표체재체IMD-pCDNA3.1(+),명명위IMD-pCDNA3.1(+),병대중조표체재체진행매절、측서감정.18지웅성Wistar대서수궤분위미전염조、공질립전염조이급IMD기인전염조,매조6지,채용초성미포개도기술전염대서신장.RT-PCR、Western blotting검측IMD표체.결과 경한제성매절감정급측서분석증실IMD-pCDNA3.1(+)재체서렬정학;3조대서간、신공능차이균무통계학의의(P균＞0.05);실험각조균미견명현신소구급신소관、간질손해;반정량RT-PCR、Western blotting검측현시:IMD전기인조신조직IMD mRNA급기단백표체교공질립전염조화미전염조명현상조.결론 IMD-pCDNA3.1(+)표체재체구건성공,병재대서신장내성공표체.
Objective To construct eukaryotic expression vector encoding rat IMD gene and deliver it into rat renal tissue via ultrasound-mircobubbles. Methods IMD gene was inserted into pCDNA3.1 ( + )between Hind Ⅲ and EcoRI enzyme sites. The recombinant plasmid designated as IMD-pCDNA 3.1 wasconfirmed by restrictive enzyme digestion and sequencing. Eighteen male Wistar rats were randomized into 3groups, which were treated with no transfection, empty vector transfection and IMD transfection, respectively, in renal tissue via ultrasound-microbubbles. RT-PCR and Western blotting were applied to detect the expression level of IMD. Results Enzyme- digestion and sequencing data showed that IMD-pCDNA 3.1 was correctly constructed. The differences in ALT, AST, BUN and SCr were not significant; No obvious damage in the glomerular, tubular and interstitial was observed in all the treated groups;Compared with non-transfection group and empty vector-transfection group, IMD mRNA and protein expression in IMD transgenic renal tissue were significantly increased. Conclusion IMD-pCDNA 3.1 expression vector was successfully constructed and well expressed in rat kidney.