粉尘螨变应原第2组分真核表达载体的构建及其在小鼠骨髓间充质干细胞中的表达  

Construction of eukaryotic expression vector for group 2 allergen of Dermatophagoides farinae and its expression in mice bone marrow mesenchymal stem cells

作　　者：卞勇华[1] 俞黎黎[1] 黄思怡[1] 史卫红[1] 崔玉宝[2] BIAN Yong-hua YU Li-li HUANG Si-yi SHI Wei-hong CUI Yu-bao(Department of Basic Medical Sciences, Yancheng Institute of Health Science, Yancheng 224005 Central Laboratory, the Third People＇s Hospital of Yancheng, Yancheng 224005, Jiangsu, China)

机构地区：[1]江苏省盐城卫生职业技术学院基础医学部,盐城市224005 [2]江苏省盐城市第三人民医院中心实验室,盐城市224005

出　　处：《广西医学》2017年第3期372-375,共4页Guangxi Medical Journal

基　　金：国家自然科学基金(31272369);江苏省自然科学基金面上项目(BK20151292);江苏省卫生厅职业技术教育研究立项课题(JZ201304);江苏省大学生实践创新训练计划一般项目(201312682011Y);江苏省盐城卫生职业技术学院青年基金专项(20114104)

摘　　要：目的构建含粉尘螨变应原第2组分(Der f 2)基因真核表达载体,并观察其在小鼠骨髓间充质干细胞(BMSCs)中的表达。方法提取粉尘螨总RNA,采用反转录聚合酶链式反应(RT-PCR)法扩增Der f 2基因,测序鉴定后将Der f 2基因克隆入pcDNA3.0质粒中,构建真核表达载体pcDNA3.0-Der f 2。将小鼠BMSCs分3组进行实验,包括pcDNA3.0-Der f2转染组、pcDNA3.0空质粒转染组及未转染组,分别利用RT-PCR、蛋白免疫印迹法(Western blot)检测Der f 2基因及蛋白在3组BMSCs中的表达。结果扩增的Der f 2基因序列与Gen Bank的参考序列完全一致,Der f 2基因正确克隆至真核表达载体pcDNA3.0中。转染BMSCs 48 h后,RT-PCR、Western blot检测结果显示,未转染组和pcDNA3.0空质粒转染组未检测到Der f 2基因表达,pcDNA3.0-Der f 2转染组检测到特异性条带,且大小与预期相符。结论 pcDNA3.0-Der f 2真核表达载体构建成功,Der f 2的mRNA和蛋白能在小鼠BMSCs中正确表达。Objective To construct eukaryotic expression vector for group 2 allergen of Dermatophagoides farina（Der f 2）,and observe its expression in mice bone marrow mesenchymal stem cells （BMSCs）.Methods Total RNA was extracted from Dermatophagoides farina,then Der f 2 gene was amplified by reverse transcription polymerase chain reaction（RT-PCR）.Der f 2 gene was cloned into pcDNA3.0 plasmid after sequencing,then eukaryotic expression vector for pcDNA3.0-Der f 2 was constructed.BMSCs of mice were divided into pcDNA3.0-Der f 2 transfection group,pcDNA3.0 transfection group,or the control group.Gene and protein expression of Der f 2 in three groups were separately detected by RT-PCR and Western blot .Results Amplified Der f 2 gene sequences were coincident with the reference sequences published in GenBank ,and Der f 2 gene was correctly cloned into eukaryotic expression vector pcDNA 3.0.After 48 hours of transfection ,the results of RT-PCR and Western blot showed that the expression of Der f 2 gene was not detected in the pcDNA 3.0 transfection group or the control group ,and specific strips were detected only in pcDNA 3.0-Der f 2 transfection group ,and the sizes of strips were coincident with the expectant sizes .Conclusion Eukaryotic expression vector of pcDNA 3.0-Der f 2 can be successfully constructed , and gene and protein of Der f 2 can express correctly in BMSCs of mice .

关 键 词：粉尘螨 变应原 第二组分 骨髓间充质干细胞 真核表达载体 基因转染 

分 类 号：R384.4[医药卫生—医学寄生虫学]