重组人Galectin-1蛋白的表达、纯化及生物活性的检测
发布时间：2018-08-02 18:42
【摘要】： Galectin-1为半乳糖凝集素(galectin)家族的重要成员之一,由135个氨基酸组成,与β-半乳糖苷具有特殊亲和力,常以单体和同源二聚体形式存在,表达于多种类型的细胞,在胸腺、平滑肌、肾脏、心肌、骨骼肌中含量丰富,具有重要的生物学功能。在神经系统中,Galectin-1参与系统发育并调节神经细胞的增殖。在免疫系统中,Galectin-1诱导胸腺细胞和活化的T细胞的凋亡从而对T细胞进行调节,并抑制炎性因子的分泌。在某些肿瘤组织中发现Galectin-1高表达,普遍参与肿瘤的发生、迁移、黏附,肿瘤血管的生成。 目前大量研究表明Galectin-1可以预防和治疗多种自身免疫性疾病,具有广阔的临床应用前景。然而,相关的动物模型实验必须给予相对高浓度的Galectin-1才能得到理想的结果。若应用于人体,则每天的剂量相当于4mg/kg,这样大的剂量无疑给Galectin-1应用于临床带来了困难。因此,如何提高Galectin-1的活性已成为其应用于临床急需解决的问题。 文献报道在体内,二聚体Galectin-1在维持免疫系统稳态过程中较单体Galectin-1具有更高的活性。而且,Patrick B?ttig等将两个Galectin-1基因串联在一起,表达的二串体重组蛋白在诱导鼠类胸腺细胞和成熟T细胞凋亡试验中比天然的单体Galectin-1表现出更强的诱导凋亡活性。因此,本课题拟采用类似设计方案,表达单体和二串体Galectin-1,筛选高活性蛋白。 为了实现上述目的,首先本课题以含有Galectin-1基因的质粒pACT-Gal-1为模板,利用PCR技术获得单体Galectin-1基因及二串体Galectin-1基因,并选择带有his标签的质粒pQE-30和本教研室改构的不含his标签的表达质粒pET-22b(+)为表达载体,通过双酶切后,将基因与表达载体相连接,构建含有Galectin-1基因的重组质粒,分别转化大肠杆菌M15和BL21(DE3)感受态细胞,构建工程菌。然后,经IPTG诱导表达,SDS-PAGE分析目的蛋白表达情况,再经Ni柱亲和层析及阴阳离子交换等不同层析方法纯化目的蛋白。最后,通过红细胞凝集试验和Jurkat细胞增殖抑制试验检测纯化所得目的蛋白的生物活性。 本课题通过对以上三方面的研究,共获得如下结果: 1)成功构建了M15/pQE-30-Gal-1、BL21/pET-22b(+)-Gal-1、BL21/pET-22b(+)-Gal-1②工程菌,双酶切鉴定和基因测序结果均正确。 2)通过Ni柱亲和层析得到纯度大于95%的His-Gal-1蛋白;通过Q阴离子交换层析和SP阳离子交换层析纯化得到纯度约为90%的Gal-1蛋白和二串体Gal-1②蛋白,Western blot结果显示均为预期目的蛋白。 3)三种重组蛋白均有红细胞凝集活性和抑制Jurkat细胞增殖活性,且二串体Gal-1②蛋白较His-Gal-1和Gal-1蛋白表现出了更强的生物活性。 综上所述,我们得到了高活性的Gal-1②重组蛋白,有望成为治疗自身免疫性疾病的候选新药。
[Abstract]:Galectin-1 is one of the important members of galactosagglutinin (galectin) family. It is composed of 135 amino acids and has special affinity with 尾 -galactoside. It often exists in the form of monomer and homologous dimer, and is expressed in many types of cells, in thymus and smooth muscle. Kidney, myocardium and skeletal muscle are abundant and have important biological functions. Galectin-1 is involved in phylogeny and regulates the proliferation of nerve cells in the nervous system. Galectin-1 induces apoptosis of thymocytes and activated T cells in the immune system, which regulates T cells and inhibits the secretion of inflammatory factors. High expression of Galectin-1 was found in some tumor tissues, which was involved in the occurrence, migration, adhesion and angiogenesis of tumor. At present, a large number of studies show that Galectin-1 can prevent and treat many autoimmune diseases, and has a broad prospect of clinical application. However, the related animal model experiments must be given a relatively high concentration of Galectin-1 in order to achieve the desired results. If applied in humans, the daily dose is equivalent to 4 mg / kg, which undoubtedly makes it difficult for Galectin-1 to be used clinically. Therefore, how to improve the activity of Galectin-1 has become an urgent problem in clinical application. In vivo, dimer Galectin-1 has higher activity than monomer Galectin-1 in maintaining immune system homeostasis. In addition, the two Galectin-1 genes were linked together by Patrick B?ttig and so on. The expressed two series of body weight histone showed stronger apoptotic activity in inducing apoptosis of mouse thymocytes and mature T cells than that of natural monomeric Galectin-1. Therefore, a similar design scheme was proposed to express monomers and distrons Galectin-1 to screen highly active proteins. In order to achieve the above purpose, the monosomic Galectin-1 gene and the distrand Galectin-1 gene were obtained by PCR using plasmid pACT-Gal-1 containing Galectin-1 gene as template. The plasmid pQE-30 with his tag and the expression plasmid pET-22b () without his tag were selected as expression vectors. The recombinant plasmid containing Galectin-1 gene was constructed by double enzyme digestion. E. coli M15 and BL21 (DE3) competent cells were transformed to construct engineering bacteria. Then the expression of the target protein was analyzed by SDS-PAGE induced by IPTG and purified by Ni column affinity chromatography and anion exchange chromatography. Finally, the biological activity of purified target protein was detected by erythrocyte agglutination test and Jurkat cell proliferation inhibition test. The results are as follows: 1) the engineering bacteria M15 / pQE-30-Gal-1 / BL21 / pET-22b () -Gal-1 / BL21 / pET-22b () -Gal-12 were successfully constructed. The results of double enzyme digestion and gene sequencing were correct. 2) the purity of His-Gal-1 protein was more than 95% by Ni column affinity chromatography. Purified by Q anion exchange chromatography and SP cation exchange chromatography, about 90% of Gal-1 protein and distring Gal-12 protein were purified by Western blot. Both of them had erythrocyte agglutination activity and inhibition of Jurkat cell proliferation. Moreover, the distron Gal-12 protein showed stronger biological activity than His-Gal-1 and Gal-1 protein. In conclusion, we have obtained highly active Gal-12 recombinant protein, which is expected to be a new drug candidate for the treatment of autoimmune diseases.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
本文编号：2160354
[Abstract]:Galectin-1 is one of the important members of galactosagglutinin (galectin) family. It is composed of 135 amino acids and has special affinity with 尾 -galactoside. It often exists in the form of monomer and homologous dimer, and is expressed in many types of cells, in thymus and smooth muscle. Kidney, myocardium and skeletal muscle are abundant and have important biological functions. Galectin-1 is involved in phylogeny and regulates the proliferation of nerve cells in the nervous system. Galectin-1 induces apoptosis of thymocytes and activated T cells in the immune system, which regulates T cells and inhibits the secretion of inflammatory factors. High expression of Galectin-1 was found in some tumor tissues, which was involved in the occurrence, migration, adhesion and angiogenesis of tumor. At present, a large number of studies show that Galectin-1 can prevent and treat many autoimmune diseases, and has a broad prospect of clinical application. However, the related animal model experiments must be given a relatively high concentration of Galectin-1 in order to achieve the desired results. If applied in humans, the daily dose is equivalent to 4 mg / kg, which undoubtedly makes it difficult for Galectin-1 to be used clinically. Therefore, how to improve the activity of Galectin-1 has become an urgent problem in clinical application. In vivo, dimer Galectin-1 has higher activity than monomer Galectin-1 in maintaining immune system homeostasis. In addition, the two Galectin-1 genes were linked together by Patrick B?ttig and so on. The expressed two series of body weight histone showed stronger apoptotic activity in inducing apoptosis of mouse thymocytes and mature T cells than that of natural monomeric Galectin-1. Therefore, a similar design scheme was proposed to express monomers and distrons Galectin-1 to screen highly active proteins. In order to achieve the above purpose, the monosomic Galectin-1 gene and the distrand Galectin-1 gene were obtained by PCR using plasmid pACT-Gal-1 containing Galectin-1 gene as template. The plasmid pQE-30 with his tag and the expression plasmid pET-22b () without his tag were selected as expression vectors. The recombinant plasmid containing Galectin-1 gene was constructed by double enzyme digestion. E. coli M15 and BL21 (DE3) competent cells were transformed to construct engineering bacteria. Then the expression of the target protein was analyzed by SDS-PAGE induced by IPTG and purified by Ni column affinity chromatography and anion exchange chromatography. Finally, the biological activity of purified target protein was detected by erythrocyte agglutination test and Jurkat cell proliferation inhibition test. The results are as follows: 1) the engineering bacteria M15 / pQE-30-Gal-1 / BL21 / pET-22b () -Gal-1 / BL21 / pET-22b () -Gal-12 were successfully constructed. The results of double enzyme digestion and gene sequencing were correct. 2) the purity of His-Gal-1 protein was more than 95% by Ni column affinity chromatography. Purified by Q anion exchange chromatography and SP cation exchange chromatography, about 90% of Gal-1 protein and distring Gal-12 protein were purified by Western blot. Both of them had erythrocyte agglutination activity and inhibition of Jurkat cell proliferation. Moreover, the distron Gal-12 protein showed stronger biological activity than His-Gal-1 and Gal-1 protein. In conclusion, we have obtained highly active Gal-12 recombinant protein, which is expected to be a new drug candidate for the treatment of autoimmune diseases.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392.1
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