Constructing and Sequencing T Vector and pET28a Expression Vector of Interleukin-2 gene

T Vector and pET28a expression vector of recombinant Interleukin-2(IL-2) were constructed and sequenced for next step work of protein expression and construction of other IL-2 fusion gene.Total RNA was prepared from human placenta,cDNA-mRNA hybrid was constructed by RT-PCR,then PCR with special primers of IL-2,the product of PCR was recycled and ligated to T vector,digested the plasmid DNA of the selected transformantion with an appropriate restriction endonuclease,the plasmid DNA was sequenced to check for mutation which is introduced by PCR.After being determined that the fragment was inserted correctly,PCR again with pfu enzyme and another primers,and the PCR fragment of IL-2 and pET28a vector were digested with two restriction endonuclease of EcoRⅠ and HindⅢ,then following the same procedure of ligating,transforming,sequencing to confirm fragment introduced into pET28a-vector.The DNA fragment introduced into the two kinds of vectors is a fragment 402 bp gene which is no signal peptide coding sequence.The sequence of IL-2 by RT-PCR from the mRNA of human single nuclear cell matches well with that in the GenBank,this result laid a sound foundation for the work of next step.