人CD40L真核表达载体构建及其在NIH3T3细胞膜上的表达

Construction of eukaryotic expression vector with gene of human CD40L and its expression in plasma membrane of NIH3T3

目的:扩增人CD40L基因并在细胞膜上表达,建立一种不需蛋白纯化获得人CD40L的简单方法,为进一步研究其在肿瘤治疗中的作用及机制奠定基础.方法:利用梯度RT-PCR从Jurkat细胞中扩增CD40L基因,测序证明序列正确后构建pcDNA3.1-40L真核表达载体,转染NIH3T3细胞,流式细胞仪检测目的基因的表达.结果:从Jurkat细胞内成功扩增人CD40L基因,筛选到一个序列无误的克隆;成功构建pcDNA3.1-40L真核表达载体;经转染NIH3T3细胞和G418初步筛选,流式细胞检测证明约41%的NIH3T3细胞膜上有CD40L基因表达.结论:利用RT-PCR技术可以从Jurkat细胞内扩增CD40L基因,利用pcDNA3.1表达载体可以实现人CD40L基因在NIH3T3细胞膜上的表达.

AIM： To amplify and express human CD40L gene in the plasma membrane of NIH3T3 for an easy and effective method to produce human CD40L protein. METHODS： Human CD40L gene was amplified by gradient RT-PCR from Jurkat cell line and subcloned into the T vector. After sequencing, pcDNA3.1- CD40L was constructed and transfected into NIH3T3 cell line. The expression of CD40L was tested by FACS. RESULTS： The CD40L gene was successfully amplified from Jurkat cell line. Sequencing identified a clone with CD40L gene without any mutation. After transfection into NIH3T3 and primary selection with G418, about 41% cells successfully transported the expressed CD40L protein to the plasma membrane. CONCLUSION： Human CD40L gene can be amplified from Jurkat cell line. pcDNA3.1 vector expresses CD40L successfully in NIH3T3 cell line and the expressed protein is partially transported to plasma membrane.