Construction of pIRES-I-A~d?? and stable expression of BALB/c mouse I-A~d?? chain genes in NIH3T3 cell line / 中国免疫学杂志

ABSTRACT

Objective:To construct pIRES-I-Ad?? bicistronic eukaryotic expression vector. Methods: Total RNA was acquired by TRIZOL method, the genes of I-Ad ?? 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 eukaiyotic 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 cytometry( FCM) was used to detennine foreign gene expression in protein level and surface expression in NIH3T3 cell line. Results: Bicistronic eukaryotic expression vector pIRES-I-AdaB was established. Foreign gene in mRNA level in transfectants was examined. Verified Ⅰ-Ad ?? chain wa3 expressed in high level expressed on transfected NIH3T3 cell line surface by FCM. Conclusion:Bicistronic eukaryotic expression vector pIRES-I-Ad ?? was constructed successfully. It is useful for studying antigen presentation and interaction between epitopes and MHC- Ⅱ molecule of BALB/c mouse.