Construction of the eukaryotic expression vectors containing different haplotypes DNA of human CIITA gene promoter IV / 重庆医科大学学报

ABSTRACT

Objective:To Construct the eukaryotic expression vectors containing four different haplotypes DNA of human CIITA gene promoter IV.Methods:Four haplotypes(CG,CC,TG and TC)can be constructed based on two single nucleotide polymorphism(SNPs)sites(G-944C and T-1350C)in promoter IV of human CIITA gene.The 487bp DNA fragments of CIITA promoter IV including the two SNPs were obtained by polymerase chain reaction(PCR)based on human genome DNA from the subjects with the CG/CG,CC/CC,TG/TG and TC/TC genotypes,which were TA cloned to pMD18-T Simple vectors and then were digested by restriction endonucleases Mlu I and Hind Ⅲ.After fragment recovery,those were ligated to four PGL3-Basic Vectors and four PGL3-Promoter Vectors respectively,which were digested by restriction endonucleases Mlu I and Hind Ⅲ as well.All recombinant plasmids were identified by sequencing.Results:Eight recombinant plasmids(CG-PGL3-Basic,CG-PGL3-Promoter,CC-PGL3-Basic,CC-PGL3-Promoter,TG-PGL3-Basic,TG-PGL3-Promoter,TC-PGL3-Basic,TC-PGL3-Promoter)containing four different haplotypes DNA of human CIITA promoter IV were obtained,whose sequences completely matched with the theoretical prediction demonstrated by sequencing.Conclusions:The eukaryotic expression vectors containing four different haplotypes DNA of human CIITA promoter IV are successfully constructed,and it lays the groundwork for the further study of different haplotype function.