Bioactivity Study Of Interleukin-7 Splice Variant Lacking Exon 4

The gene of Interleukin-7(IL-7) includes six exons, one or more of these exons is skipped when affected by some unknown factors, leading to many m RNA of different lengths that coming from the same gene, a series of Interleukin-7 splice variants appear as a result. IL-7δ4, IL-7 splice variant lacking exon 4, whose primary structure differs from IL-7, may participate in IL-7’s function adjustment, and even gain new functions or antagonistic functions. IL-7δ4m RNA can be detected in many normal human tissues and organs, but its physiology has not been addressed up to now. In addition, its m RNA expression is abnormally increased in several tumor cell lines and tissues and organs of patients with multiple sclerosis, whether it is related to the occurrence and development of diseases also needs to be researched. Objective:In this paper, we use the prokaryotic expression vector p ET21 b to express rh IL-7δ4, the recombinant protein was refolded and purified after its expression, and identified with Western Blot, then preliminarily study its bioactivity, which will lay the foundation for its further functional study. Its further functional study has very important significance on probe into IL-7’s function and regulation and helps to timely diagnosis and therapy related diseases. Methods:1. Preparation of rh IL-7δ4: Use 18T- IL-7δ4 that maintained in our lab as the template to carry out the TA cloning, then to construct a prokaryotic expression vector p ET-21b-IL-7δ4, following the expression and soluble analysis of rh IL-7δ4 and optimizing its expression conditions. Recombinant protein was refolded and purified after its expression, then identified with Western Blot.2. The effect of rh IL-7δ4 on the peripheral blood mononuclear cells: After measured its concentration by BCA method, the purified protein was targeted to the peripheral blood mononuclear cells(PBMCs), followed by analyzing its BCL-2 protein expression and apoptosis rates in response to rh IL-7δ4 on the base of taking rh IL-7 as positive control.3. The effect of rh IL-7δ4 on the lung fibroblasts MRC-5: The proliferation of MRC-5 cells observed by MTT method when stimulated with TGF-β1, rh IL-7and rh IL-7δ4 respectively, or TGF-β1 and rh IL-7, or TGF-β1 and rh IL-7δ4, and western blot analysised its collagen I and α-SMA protein expression. Restuls:1. Expressed, refolded and purified rh IL-7δ4: Recombinant p ET-21b-IL-7δ4 of the prokaryotic expression vector was constructed successfully, Amplified fragment sizes by PCR were equal to IL-7-δ4(399bp) that expected and its results of DNA sequencing were as the same as that of Gen Bank reported. Its refolding efficiency of IL-7δ4 was about 65%, and its purity was﹥95% after purification. What’s more, Western Blot proved that the expressed product was rh IL-7δ4 protein.2. The effect of rh IL-7δ4 on the human PBMCs: In vitroe, higher BCL-2 protein and lower apoptosis rates in response to rh IL-7δ4 compared with the control(P<0.01), that was as the same as rh IL-7.3. The effect of rh IL-7δ4 on the MRC-5s: Cell proliferation improved when stimulated with TGF-β1, rh IL-7 and rh IL-7δ4 respectively(P<0.01), but there was difference among them(TGF-β1> rh IL-7δ4> rh IL-7). What’s more, all of them can induce the cells to expresse the collagen I and α-SMA protein(P<0.05). When rh IL-7or rh IL-7δ4 combined with TGF-β1, the increase were more significant. However, if prior to use IL-7R antibody to block the IL7 R of the cells, the cell protein expression significantly decreased(P<0.05). Conclusion:In the present study, we express rh IL-7-δ4 with prokaryotic expression system and have determined its best expression parameters, rh IL-7δ4 with high purity and bioactivity was successfully gained via refolding and purifying. It can upgrade BCL-2 protein expressions in PBMCs and restrain its apoptosis, which means that rh IL-7δ4 was also involved in the immune regulation. What’s more, rh IL-7δ4 can promote MCR-5 proliferation and up regulate its collagen I and α-SMA protein expression, suggesting that rh IL-7δ4 promotes fibroblasts.