| Prothymosinα(proTα)is an acidic protein which can promote cell proliferation and will be of a significant immunostimulating activity. It has been used in the treatment of cancers, hepatitis and immune system diseases. Interleukin 2( IL-2) is a glycoprotein that can enhance the activity of NK cells.IL-2 plays a significant effect on curing renal cell carcinoma, melanoma, colon and rectal cancer. Whereas, patients will have a side-effect with excessive IL-2. ProTαand IL-2 can synergize on stimulating the immune system and proTαcan reduce the effective concentration of IL-2 by half throug regulating the activity of NK sensitive cell and LAK cell toxicity induced by IL-2. ProTαand IL-2 have a stronger effect on the treatment of autologous tumor than IL-2 alone. ProTαand IL-2 used together can not only enhance the anti-tumor effects ,but also decrease the toxicity produced by IL-2 alone.In this reseach, proTα-linker-IL-2 fusion gene was cloned from pcDNA3.1-PTI plasmid by PCR technique.It was modified by adding a 51 nt sequence coded for a seventeen amino acid GM-CSF signal peptide at the 5'-end. The modified proTα-linker-IL-2 fusion gene was then cloned into eukaryotic expressing vector pVAX and the recombinant plasmid pVAX-PI was transformed into DH5α. The plasmid was extracted from the positive bacteria for restriction endonuclease analysis and DNA sequencing. The sequencing result showed that the sequence of the modified fusion gene was coincided with the expected.The endotoxin free recombinant plasmid pVAX-PI was extracted and quantitative analysed. Then the recombinant plasmid was transfected into COS-7 cells in vitro with transfection reagent lipofectamine and the pVAX-PI plasmid could express in eukaryotic cells at anytime after COS-7 transfection. The expression quantity of IL-2 in the supernatant was anayzed by ELISA at 24 h,48 h,72 h and 96 h post-transfection and the concentration of IL-2 protein was about 5.4,7.8,6.1,5.7 ug/L.The biological activity of IL-2 in the supernatant at 24 h,48 h,72 h and 96 h after transfection was 43,58,54,49 IU/ml assayed by CTLL-2 cell line proliferation. And the culture supernatant could obviously stimulate the proliferation of mice splenocytes. The molecular weight of the fusion protein(31kDa) was analyzed and identified by ECL western blotting.In this research,pVAX-PI plasmid had an anti-tumor activity to melanoma tumor and could induce the tumor-bearing mice to produce cytokines of IFN-γand IL-4. The quantity of IFN-γand IL-4 in the peripheral blood of the tumor-bearing mice immunized with pVAX-PI plasmid was gradually increased. Five weeks after immunization, IFN-γand IL-4 were 222 pg/ml and 157 pg/ml in the peripheral blood of the tumor-bearing mice inoculated by intramuscular combined with electric-transfection and were 129 pg/ml and 68 pg/ml in the peripheral blood of the tumor-bearing mice inoculated by intramuscular respectively. The above data showed that pVAX-PI plasmid had an effect on immune regulation system in tumor-bearing mice. |
