重組人Galectin-1蛋白的表達(dá)、純化及生物活性的檢測(cè)
發(fā)布時(shí)間：2018-08-02 18:42
【摘要】： Galectin-1為半乳糖凝集素(galectin)家族的重要成員之一,由135個(gè)氨基酸組成,與β-半乳糖苷具有特殊親和力,常以單體和同源二聚體形式存在,表達(dá)于多種類型的細(xì)胞,在胸腺、平滑肌、腎臟、心肌、骨骼肌中含量豐富,具有重要的生物學(xué)功能。在神經(jīng)系統(tǒng)中,Galectin-1參與系統(tǒng)發(fā)育并調(diào)節(jié)神經(jīng)細(xì)胞的增殖。在免疫系統(tǒng)中,Galectin-1誘導(dǎo)胸腺細(xì)胞和活化的T細(xì)胞的凋亡從而對(duì)T細(xì)胞進(jìn)行調(diào)節(jié),并抑制炎性因子的分泌。在某些腫瘤組織中發(fā)現(xiàn)Galectin-1高表達(dá),普遍參與腫瘤的發(fā)生、遷移、黏附,腫瘤血管的生成。 目前大量研究表明Galectin-1可以預(yù)防和治療多種自身免疫性疾病,具有廣闊的臨床應(yīng)用前景。然而,相關(guān)的動(dòng)物模型實(shí)驗(yàn)必須給予相對(duì)高濃度的Galectin-1才能得到理想的結(jié)果。若應(yīng)用于人體,則每天的劑量相當(dāng)于4mg/kg,這樣大的劑量無(wú)疑給Galectin-1應(yīng)用于臨床帶來(lái)了困難。因此,如何提高Galectin-1的活性已成為其應(yīng)用于臨床急需解決的問(wèn)題。 文獻(xiàn)報(bào)道在體內(nèi),二聚體Galectin-1在維持免疫系統(tǒng)穩(wěn)態(tài)過(guò)程中較單體Galectin-1具有更高的活性。而且,Patrick B?ttig等將兩個(gè)Galectin-1基因串聯(lián)在一起,表達(dá)的二串體重組蛋白在誘導(dǎo)鼠類胸腺細(xì)胞和成熟T細(xì)胞凋亡試驗(yàn)中比天然的單體Galectin-1表現(xiàn)出更強(qiáng)的誘導(dǎo)凋亡活性。因此,本課題擬采用類似設(shè)計(jì)方案,表達(dá)單體和二串體Galectin-1,篩選高活性蛋白。 為了實(shí)現(xiàn)上述目的,首先本課題以含有Galectin-1基因的質(zhì)粒pACT-Gal-1為模板,利用PCR技術(shù)獲得單體Galectin-1基因及二串體Galectin-1基因,并選擇帶有his標(biāo)簽的質(zhì)粒pQE-30和本教研室改構(gòu)的不含his標(biāo)簽的表達(dá)質(zhì)粒pET-22b(+)為表達(dá)載體,通過(guò)雙酶切后,將基因與表達(dá)載體相連接,構(gòu)建含有Galectin-1基因的重組質(zhì)粒,分別轉(zhuǎn)化大腸桿菌M15和BL21(DE3)感受態(tài)細(xì)胞,構(gòu)建工程菌。然后,經(jīng)IPTG誘導(dǎo)表達(dá),SDS-PAGE分析目的蛋白表達(dá)情況,再經(jīng)Ni柱親和層析及陰陽(yáng)離子交換等不同層析方法純化目的蛋白。最后,通過(guò)紅細(xì)胞凝集試驗(yàn)和Jurkat細(xì)胞增殖抑制試驗(yàn)檢測(cè)純化所得目的蛋白的生物活性。 本課題通過(guò)對(duì)以上三方面的研究,共獲得如下結(jié)果: 1)成功構(gòu)建了M15/pQE-30-Gal-1、BL21/pET-22b(+)-Gal-1、BL21/pET-22b(+)-Gal-1②工程菌,雙酶切鑒定和基因測(cè)序結(jié)果均正確。 2)通過(guò)Ni柱親和層析得到純度大于95%的His-Gal-1蛋白;通過(guò)Q陰離子交換層析和SP陽(yáng)離子交換層析純化得到純度約為90%的Gal-1蛋白和二串體Gal-1②蛋白,Western blot結(jié)果顯示均為預(yù)期目的蛋白。 3)三種重組蛋白均有紅細(xì)胞凝集活性和抑制Jurkat細(xì)胞增殖活性,且二串體Gal-1②蛋白較His-Gal-1和Gal-1蛋白表現(xiàn)出了更強(qiáng)的生物活性。 綜上所述,我們得到了高活性的Gal-1②重組蛋白,有望成為治療自身免疫性疾病的候選新藥。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
本文編號(hào)：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 郭廣君;呂素芳;王榮富;;外源基因表達(dá)系統(tǒng)的研究進(jìn)展[J];科學(xué)技術(shù)與工程;2006年05期
2 牛耀輝;陳朝銀;趙聲蘭;楊蘇;唐嘉;;凝集素及其抗HIV研究進(jìn)展[J];云南化工;2006年06期
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