关键词: 人血清白蛋白基因, 载体, 序列鉴定

Abstract: Objective To construct and sequence T Vector and pET₂8a expression vector of recombinant Human Serum Albumin(rHSA) for protein expression and construction of other rHSA fusion gene.Methods Total RNA was prepared from human placenta,cDNA-mRNA hybrid was constructed by RT-PCR,then PCR with special primers of rHSA,the product of PCR was recycled and ligated to T vector,after the procedure of transforming an aliquot of each ligation into E.coli DH-5α and digesting the plasmid DNA of the selected transformants with an appropriate restriction endonuclease,the plasmid DNA was sequenced to check for the information of the inserted fragment.With the corrected fragment of rHSA,PCR again with pfu enzyme and another primers,and the PCR fragment and pET₂8a vector were digested with two restriction endonucleases of EcoRⅠ and HindⅢ,then followed the same procedure of ligating,transforming,sequencing to confirm fragment introduced into pET₂8a-vector.Results The DNA fragment introduced into the two kinds of vectors is a fragment 1 830 bp gene which includes 72 bp signal peptide coding sequence,it shows the possibility of gene polymorphism at alleles 485,750,1 685.Conclusion The sequence of rHSA by RT-PCR from the mRNA of placenta matches well with that in the GenBank,this result can lay a solid foundation for the work of next step.

Key words: recombinant human serum albumin(rHSA), vector, sequencing