Cloning Of The Human Gene For Prothymosin-Interleukin Fusion Protein And Constructing Of Its Eukaryotic Expression Vector

Human prothymosin α( ProT α) is a highly acidic protein which is distributed widely and is high conserved among mammals. It is a fact that ProT α has many immunomodulatory functions which may be of great benefit to clinical therapy.The six members of the gene family have been cloned and sequenced. The ProT α gene is located at chromosome 2, Its molecular weight is 13 kDa. A ProT α gene of Caucasian has been cloned and its expression product is undertaking clinical trails to tumor patients.Interleukin-2 (IL2), formerly referred to as T-cell growth factor, is a powerfully immunoregulatory lymphokine that is produced by lectin- or antigen-activated T cells. IL-2 gene is located at chromosome 4 and its molecular weight is 15 kDa. Not only is it produced by mature T lymphocytes on stimulation but also constitutively by certain T-cell lymphoma cell lines. It is useful in the study of the molecular nature of T-cell differentiation and because, it augments natural killer cell activity, it might have use in the treatment of cancer. But if we overuse the IL-2,it can bring about bad effect.The combination of IL-2 and prothymosin alpha (ProTa) resulted in a greater response to the autologous tumor than that brought about by IL-2 alone. The combination of IL-2 and ProTα can not only decrease toxiferous function of overuse IL-2,but also increase anti-tumor effect.Based on the nucleotide sequence of ProT αand IL-2 published in Genbank,two pairs of RT-PCR primers were designed and synthesized.Total human T cell and HL-60 leukemia cell RNA were isolated and used as template to generate complementary of DNA ProT αand IL-2 by reverse transcription. Then the primers with linkers were designed.Two sigle genes were linked with linker and combined to a ProTα-IL-2 gene.we cloned the ProTα and ProTα-IL-2 into pcDNA3.1(-) plasmid.DNA sequence analysis showed that the cloned sequence of ProTα and IL-2 have very high similarity with the sequence of ProTα and IL-2 published in Genbank.