双重抗肺纤维化基因嵌合重组体蛋白的研制
发布时间：2018-11-25 12:32
【摘要】： 目的:中性粒细胞抑制因子(NIF)是整合素β2的专一性拮抗剂,能抑制中性粒细胞与内皮细胞的粘附和聚集,阻止白细胞的渗透和浸润。角质细胞生长因子(KGF)是成纤维细胞生长因子家族中的一员,由间充质细胞分泌并特异性地作用于上皮细胞促进其生长繁殖。许多实验均证明KGF和NIF在肺脏的发育、炎症和修复中发挥重要的作用。针对NIF能够抑制病理性自身免疫反应和KGF能够修复损伤的肺泡上皮及抑制纤维化的功效,我们构建了这两种细胞因子的基因嵌合重组体,并进行表达,目的是研制能够治疗急性肺损伤和肺纤维化的重组蛋白,为进一步的研究和开发奠定基础。方法:以8个甘氨酸作为铰链区,运用over-lap PCR的方法将NIF与KGF连接,获得双重抗肺纤维化基因嵌合重组体NKM的基因序列。利用pET原核表达系统,在BL21 Star (DE3)plysS大肠杆菌中高效表达NKM。经过测序、诱导表达、纯化和Western-Blot等确证后,再对NKM的体内外活性进行测定。结果:本研究成功构建了双重抗肺纤维化基因重组嵌合体NKM;并在BL21 Star (DE3)plysS大肠杆菌中表达量达到25%;经Western-Blot检测有免疫活性;通过DEAE层析柱纯化得到纯度为80%的NKM;体外细胞活性测定表明,NKM对白细胞粘附有剂量依赖性的抑制作用,对成纤维细胞生长也有剂量依赖性的抑制作用;动物体内活性测定显示,NKM抑制急性肺损伤后循环内白细胞的增加;病理解剖显示NKM能明显抑制博莱霉素诱导的肺炎和肺纤维化。结论:本实验获得了具有双重功能的抗肺纤维化嵌合体蛋白,可用于治疗急性肺损伤和肺纤维化,并有望用于治疗其它与白细胞活化浸润及组织纤维化有关的多种临床疾病。
[Abstract]:Aim: (NIF) is a specific antagonist of integrin 尾 2, which can inhibit the adhesion and aggregation of neutrophils to endothelial cells and prevent the infiltration and infiltration of leukocytes. Keratinocyte growth factor (KGF) is a member of fibroblast growth factor family, secreted by mesenchymal cells and acting specifically on epithelial cells to promote their growth and reproduction. Many experiments have demonstrated that KGF and NIF play an important role in lung development, inflammation and repair. In view of the inhibition of pathological autoimmune response by NIF and the ability of KGF to repair damaged alveolar epithelium and inhibit fibrosis, we constructed and expressed the chimeric recombinant of these two cytokines. The aim is to develop recombinant protein which can treat acute lung injury and pulmonary fibrosis and lay the foundation for further research and development. Methods: using 8 glycine as hinge regions, NIF and KGF were connected by over-lap PCR to obtain the gene sequence of double anti-pulmonary fibrosis gene chimeric recombinant NKM. Expression of NKM. in BL21 Star (DE3) plysS Escherichia coli using pET prokaryotic expression system After sequencing, induced expression, purification and Western-Blot, the activity of NKM in vivo and in vitro was determined. Results: in this study, double anti-pulmonary fibrosis gene recombinant chimeric NKM; was successfully constructed and expressed in BL21 Star (DE3) plysS Escherichia coli. The cell activity of NKM; with purity of 80% was determined by DEAE column purification in vitro. The results showed that NKM inhibited leukocyte adhesion in a dose-dependent manner and the growth of fibroblasts in a dose-dependent manner. In vivo, NKM inhibited the increase of leukocyte in circulation after acute lung injury, and the pathological anatomy showed that NKM could inhibit bleomycin induced pneumonia and pulmonary fibrosis. Conclusion: this study has obtained double function anti-pulmonary fibrosis chimeric proteins which can be used in the treatment of acute lung injury and pulmonary fibrosis and may be used in the treatment of other clinical diseases related to leukocyte activation infiltration and tissue fibrosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
本文编号：2356117
[Abstract]:Aim: (NIF) is a specific antagonist of integrin 尾 2, which can inhibit the adhesion and aggregation of neutrophils to endothelial cells and prevent the infiltration and infiltration of leukocytes. Keratinocyte growth factor (KGF) is a member of fibroblast growth factor family, secreted by mesenchymal cells and acting specifically on epithelial cells to promote their growth and reproduction. Many experiments have demonstrated that KGF and NIF play an important role in lung development, inflammation and repair. In view of the inhibition of pathological autoimmune response by NIF and the ability of KGF to repair damaged alveolar epithelium and inhibit fibrosis, we constructed and expressed the chimeric recombinant of these two cytokines. The aim is to develop recombinant protein which can treat acute lung injury and pulmonary fibrosis and lay the foundation for further research and development. Methods: using 8 glycine as hinge regions, NIF and KGF were connected by over-lap PCR to obtain the gene sequence of double anti-pulmonary fibrosis gene chimeric recombinant NKM. Expression of NKM. in BL21 Star (DE3) plysS Escherichia coli using pET prokaryotic expression system After sequencing, induced expression, purification and Western-Blot, the activity of NKM in vivo and in vitro was determined. Results: in this study, double anti-pulmonary fibrosis gene recombinant chimeric NKM; was successfully constructed and expressed in BL21 Star (DE3) plysS Escherichia coli. The cell activity of NKM; with purity of 80% was determined by DEAE column purification in vitro. The results showed that NKM inhibited leukocyte adhesion in a dose-dependent manner and the growth of fibroblasts in a dose-dependent manner. In vivo, NKM inhibited the increase of leukocyte in circulation after acute lung injury, and the pathological anatomy showed that NKM could inhibit bleomycin induced pneumonia and pulmonary fibrosis. Conclusion: this study has obtained double function anti-pulmonary fibrosis chimeric proteins which can be used in the treatment of acute lung injury and pulmonary fibrosis and may be used in the treatment of other clinical diseases related to leukocyte activation infiltration and tissue fibrosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392
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