BALB/c小鼠I-A^dαβ链编码基因真核双顺反子载体的构建及表达  

Construction of pIRES-I-A^dαβ and stable expression of BALB/c mouse I-A^dαβ chain genes in NIH3T3 cell line

作　　者：高明[1] 王海平[1] 周勇[1] 王全立[1] 

机构地区：[1]军事医学科学院野战输血研究所,北京100850

出　　处：《中国免疫学杂志》2006年第1期41-43,共3页Chinese Journal of Immunology

基　　金：国家自然科学基金资助(30200245)

摘　　要：目的:为了获取BALB/c小鼠I-Adα、β链编码基因,构建真核双顺反子表达载体并在NIH3T3细胞中表达,建立BALB/c小鼠MHC-Ⅱ抗原递呈细胞研究模型。方法:采用Trizol提取BALB/c小鼠脾细胞总RNA,RT-PCR获取I-Adαβ链 cDNA,连接pGEMT载体测序,正确后亚克隆至pIRES真核双顺反子表达载体;用Lipofectamine2000转染NIH3T3细胞,G418 筛选转染克隆株。RT-PCR鉴定外源基因在mRNA水平的表达;流式细胞术(FCM)鉴定外源基因在蛋白质水平的表达以及在细胞表面展示的水平。结果:建立了BALB/c小鼠I-Adαβ链编码基因真核双顺反子表达载体pIRES-I-Adαβ;pIRES-I-Adαβ转染NIH3T3细胞。在G418筛选下可获得高达72％的转染细胞;转染细胞总RNA RT-PCR显示外源基因在mRNA水平得到表达;FCM显示外源基因编码的蛋白高水平表达并定位于细胞表面。结论:为进一步研究BALB/c小鼠MHC-Ⅱ抗原递呈的分子机制奠定了基础。Objective：To construct plRES-I-A^d αβ bicistronic eukaryotic expression vector. Methods：Total RNA was acquired by TRIZOL method, the genes of I-A^d αβ chains were amplified through RT-PCR, respectively. The target genes were connected to pGEM-T vector and sequenced. Then the target genes were subcloned into pIRES bicistronic eukaryotic expression vector and NIH3T3 cell line was transfected. Transfectant was screened by G418 antibiotics. Total RNA of transfectant was obtained by TRIZOL method, mRNA of foreign gene was examined by RT-PCR. Flow eytometry（FCM） was used to determine foreign gene expression in protein level and surface expression in NIH3T3 cell line. Results： Bicistronic eukaryotic expression vector pIRES-I-A^d αβ was established. Foreign gene in mRNA level in transfectants was examined. Verified I-A^d αβ chain was expressed in high level expressed on transfected NIH3T3 cell line surface by FCM. Conclusion：Bicistronic eukaryotic expression vector pIRES-I-A^d αβ was constructed successfully, It is useful for studying antigen presentation and interaction between epitopes and MHC-Ⅱ molecule of BALB/c mouse.

关 键 词：抗原递 BALB/C小鼠 I-A^d αβ链 转染 NIH3T3细胞 

分 类 号：R392.2[医药卫生—免疫学]