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白细胞介素10(IL-10)是一种多效细胞因子,在炎症、免疫反应以及在疾病的发生过程中发挥着重要作用,RGD是能够特异与新生血管内皮细胞整合素结合的多肽序列。将RGD连接到IL-10的羧基端,期望构建新生血管内皮细胞特异导向结合型融合蛋白。以人外周血淋巴细胞cDNA为模板,扩增的PCR产物经克隆载体pMD18-T,连至原核表达载体pET-22b(+),转化大肠杆菌BL21(DE3),构建了pET-IL10-RGD表达载体的重组菌(pET-IL10-RGD/BL21)。SDS-PAGE分析表明:在19.3 kDa处有明显的新生蛋白带,符合理论预期。Western blot分析表明:诱导表达、分离纯化的目的蛋白能够与IL-10抗体特异结合,且纯化产物IL10-RGD具有与IL-10相同的生物学活性。利用培养的人皮肤成纤维细胞,观察了IL10-RGD对TGF-β1刺激成纤维细胞的I型胶原(Col1)、III型胶原(Col3)、α-平滑肌肌动蛋白(α-SMA)、结体组织生长因子(CTGF)蛋白水平及α-SMA免疫细胞化学的变化。结果表明:纯化产物IL10-RGD能够明显抑制TGF-β1刺激的成纤维细胞Col1、Col3、CTGF和α-SMA蛋白水平的升高;抑制TGF-β1诱导的成纤维细胞向肌成纤维细胞的转化。可见,成功克隆、表达并纯化了IL10-RGD融合蛋白,该融合蛋白能够明显拮抗TGF-β1诱导的纤维化,预示着该蛋白在瘢痕增生及皮肤纤维化治疗方面有着较好的应用前景。
Interleukin-10 (IL-10) is a pleiotropic cytokine that plays an important role in inflammation, immune response and in the pathogenesis of diseases. RGD is a polypeptide sequence that can specifically bind to integrin of neovascular endothelial cells. Connecting RGD to the carboxy terminus of IL-10, it is desirable to construct a neovascular endothelial cell that specifically targets a binding fusion protein. The PCR product of human peripheral blood lymphocyte cDNA was cloned into the prokaryotic expression vector pET-22b (+) by cloning vector pMD18-T and transformed into E.coli BL21 (DE3) to construct the expression vector pET-IL10-RGD Vector of recombinant bacteria (pET-IL10-RGD / BL21). SDS-PAGE analysis showed that at 19.3 kDa obvious neo-protein band, in line with the theoretical expectation. Western blot analysis showed that IL-10 could bind specifically with IL-10 antibody after induction and expression, and the purified product IL10-RGD had the same biological activity as IL-10. The cultured human skin fibroblasts were used to observe the effect of IL10-RGD on the expression of Col1, Col3, α-SMA and TGF-β1-stimulated fibroblasts. Changes of Tissue Growth Factor (CTGF) Protein Levels and α-SMA Immunocytochemistry. The results showed that the purified product IL10-RGD could significantly inhibit the TGF-β1-stimulated fibroblasts Col1, Col3, CTGF and α-SMA protein levels increased; TGF-β1-induced fibroblast transformation to myofibroblasts . It can be seen that the IL10-RGD fusion protein was cloned, expressed and purified successfully, and the fusion protein can obviously antagonize the TGF-β1-induced fibrosis, indicating that the protein has good application prospect in the treatment of scar hyperplasia and skin fibrosis.