中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2012年
10期
1338-1341
,共4页
徐洪涛%杨慧%常立文%李文斌%赵玲霞%袁文浩%刘伟%蔡保欢%王席娟
徐洪濤%楊慧%常立文%李文斌%趙玲霞%袁文浩%劉偉%蔡保歡%王席娟
서홍도%양혜%상립문%리문빈%조령하%원문호%류위%채보환%왕석연
支气管肺发育不良/遗传学%基因表达%遗传载体%肺表面活性物质相关蛋白质B/遗传学
支氣管肺髮育不良/遺傳學%基因錶達%遺傳載體%肺錶麵活性物質相關蛋白質B/遺傳學
지기관폐발육불량/유전학%기인표체%유전재체%폐표면활성물질상관단백질B/유전학
Bronchopulmonary dysplasia/genetics%Gene expression%Genetic vectors%Pulmonary surfactant-associated protein B/genetics
目的 构建两种人肺表面活性物质蛋白B(surfactant protein B,SP-B)基因变异型的真核表达载体,研究SP-B基因多态性与支气管肺发育不良(bronchopulmonary dysplasia,BPD)的关系.方法 应用基因重组技术,构建pIRES2-EGFP-SP-B-C 1580和pIRES2-EGFP-SP-B-T 1580真核表达质粒,经测序鉴定后,采用脂质体法转染293T细胞系,荧光显微镜观察绿色荧光蛋白在293T细胞中的表达,逆转录-聚合酶链反应-限制性片段长度多态性(reverse transcription-polymerase chain reaction-restriction fragment length polymorphism,RT-PCR-RFLP)检测SP-B-C/T 1580 mRNA的表达,Westem blot 检测SP-B蛋白的表达.结果 两个重组质粒含有完整SP-B cDNA,在1580位点碱基序列分别为C和T,转染293T细胞后有相应的mRNA及蛋白表达.结论 成功构建了真核表达载体pIRES2-EG-FP-SP-B-C/T 1580,为揭示支气管肺发育不良的发生机制奠定基础.
目的 構建兩種人肺錶麵活性物質蛋白B(surfactant protein B,SP-B)基因變異型的真覈錶達載體,研究SP-B基因多態性與支氣管肺髮育不良(bronchopulmonary dysplasia,BPD)的關繫.方法 應用基因重組技術,構建pIRES2-EGFP-SP-B-C 1580和pIRES2-EGFP-SP-B-T 1580真覈錶達質粒,經測序鑒定後,採用脂質體法轉染293T細胞繫,熒光顯微鏡觀察綠色熒光蛋白在293T細胞中的錶達,逆轉錄-聚閤酶鏈反應-限製性片段長度多態性(reverse transcription-polymerase chain reaction-restriction fragment length polymorphism,RT-PCR-RFLP)檢測SP-B-C/T 1580 mRNA的錶達,Westem blot 檢測SP-B蛋白的錶達.結果 兩箇重組質粒含有完整SP-B cDNA,在1580位點堿基序列分彆為C和T,轉染293T細胞後有相應的mRNA及蛋白錶達.結論 成功構建瞭真覈錶達載體pIRES2-EG-FP-SP-B-C/T 1580,為揭示支氣管肺髮育不良的髮生機製奠定基礎.
목적 구건량충인폐표면활성물질단백B(surfactant protein B,SP-B)기인변이형적진핵표체재체,연구SP-B기인다태성여지기관폐발육불량(bronchopulmonary dysplasia,BPD)적관계.방법 응용기인중조기술,구건pIRES2-EGFP-SP-B-C 1580화pIRES2-EGFP-SP-B-T 1580진핵표체질립,경측서감정후,채용지질체법전염293T세포계,형광현미경관찰록색형광단백재293T세포중적표체,역전록-취합매련반응-한제성편단장도다태성(reverse transcription-polymerase chain reaction-restriction fragment length polymorphism,RT-PCR-RFLP)검측SP-B-C/T 1580 mRNA적표체,Westem blot 검측SP-B단백적표체.결과 량개중조질립함유완정SP-B cDNA,재1580위점감기서렬분별위C화T,전염293T세포후유상응적mRNA급단백표체.결론 성공구건료진핵표체재체pIRES2-EG-FP-SP-B-C/T 1580,위게시지기관폐발육불량적발생궤제전정기출.
Objective To construct two kinds of eukaryotic ccll expression vcctors pIRES2-EGFP-SP-B-C/T 1580 and evaluate their expressions in 293T cells,for the further study of relationship between polymorphism of surfactant protein B (SP-B) gene and bronchopulmonary dysplasia (BPD).Methods The eukaryotic pIRES2-EGFP-SP-B-C/T 1580 expression vectors were constructed by gene recombination,and identified by gene sequencing.The recombinant expression vectors were transfected into 293T cells by lipofectamine2000.The expression of green fluorescence protein in 293T cells was observed by fluorescence microscopy.The mRNAs and proteins of SP-B-C/T 1580 were tested and identified by reverse transcriptionpolymerase chain reaction-restriction fragment length polymorphism(RT-PCR-RFLP) and western blot.Results Two recombinant plasmids contained the complete cDNA of SP-B with the same sequence as in gene bank.The base of SP-B 1580 gene of pIRES2-EGFP-SP-B-C 1580 was C,that of pIRES2-EGFP-SP-B-T 1580 was T.After being transfected into 293T cells,highly efficient expression of SP-B-C/T 1580 gene was detected at mRNA and protein levels.Conclusions The pIRES2-EGFP-SP-B-C/T 1580 eukaryotic cell expression vectors were successfully constructed.
