人参愈伤组织细胞表达CTB-hIFN-α2b融合蛋白的研究
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摘要
人干扰素α2b(hIFN-α2b)具有多种生物学功能,包括广谱抗病毒、抑制肿瘤细胞增殖和免疫调节的作用,在临床上主要用于治疗病毒性肝炎及肿瘤性疾病。目前,临床应用的干扰素制剂主要是从大肠杆菌表达系统获得的基因工程重组蛋白,并以注射给药为主。由于干扰素在血液中的半衰期短,静脉注射后很快从血中消失,故很难维持较高的血药浓度,在临床应用上只能以提高用药频率和用药量来解决,这样就很容易诱发干扰素抗体的产生及增加毒副作用。因此,如何改进给药途径是其开发的一个重要方向。霍乱毒素B亚单位(CTB)是霍乱毒素的无毒结合亚基,具有载体蛋白的特性,并且由于CTB本身具有很强的免疫原性,能剌激血液和肠道产生特异性保护抗体,与其他抗原共同使用还具有免疫佐剂的作用,能增强抗原蛋白的免疫效力和免疫特异性。人参是一种珍贵的药用植物,具有独特的营养和药用价值。近年来,利用转基因植物作为生物反应器生产药用蛋白正得以深入研究,多方面的报道证明植物可以高水平表达具有功能的重组蛋白质。
在本研究中,我们构建了含有霍乱毒素B亚单位与人干扰素α2b融合基因片段的植物细胞表达载体,选择人参愈伤组织细胞作为生物反应器,利用农杆菌介导的转化法将融合基因导入人参愈伤组织细胞,得到了能够同时表达hIFN-α2b和CTB的转基因人参愈伤组织细胞系。为建立以人参愈伤组织细胞作为生物反应器生产口服药用蛋白奠定了基础,并为药用蛋白和药用植物的联合应用开辟了一条新思路。
Human interferonα2b(hIFN-α2b)processes a variety of biological functions, including broad-spectrum antiviral, anti-cell division and immunomodulatory activities. In the clinic, it′s mainly for the treatment of viral hepatitis and cancer diseases. At present, the clinical application of interferon preparations are mainly obtained from the E.coli expression system, and the main administration is injection. Because the half-life is short, interferon would soon disappeare from the blood after the intravenous injection. It is difficult to maintain a high plasma concentration in clinical applications. The problem can only be solved through increasing the frequency and dosage. So that it is easy to produce interferon-induced antibody and increase the toxic side effects. The injected administration is inconvenient and expensive for patients. Therefore, it was need to develop new formulations of interferon.
Cholera toxin B subunit (CTB) is a non-toxic binding subunit of cholera toxin, it can specific bind with the gangliosides GM1 receptor which placed on the membrane of nucleated cells. CTB can be used for carrier protein, so exogenous peptides may be combined with CTB in order to increase the opportunity to contact with the cell membrane receptor. Based on these characteristics of CTB, today it has been applied to the oral vaccines and oral drugs research.
In recent years, the use of transgenic plants producing pharmaceutical proteins are able to in-depth study. Compared with the traditional system, the plant expression system is efficient, economical and convenient and so on. In addition, the most important edible plants also have characteristics. Therefore, the use of transgenic plants as bioreactors production of oral pharmaceutical proteins are being increasingly subject to national attention. It was report that plants can express high levels of functional recombinant proteins.
For transgenic plant material can be divided into plants, explant and callus cells, and so on. Plant tissue cell culture studies have decades of history. Compared with conventional agricultural production, it is not impacted by the ecological environment and the pests. Ginseng is a precious medicinal plants, has a unique nutritional and medicinal value. The common receptors of transgenic plants are tobacco, rice, tomato, potato and so on. The expression of human interferon in ginseng has not been reported.
In this study, human interferonα2b and cholera toxin B subunit gene was fused by gene fusion, which CTB was located at the N-terminal and hIFN-α2b was placed at the C-terminal. They were linked by a flexible peptide chain. First, the fusion gene was constructed into the pMD18-T vector to form the pMDCI. The pMDCI and pBI121 was digested with BamH I and Sac I. The target gene fragment and vector fragments were recovered by agarose gel electrophoresis and connected to receive expression vector. The recombinant plasmid was confirmed by PCR and restriction digestion, finally obtaining the plant expression vector pBICI.
The pBICI was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBICI was used to transform ginseng cells. Ginseng cells were co-cultivated for 48～72 h with Agrobacterium tumefaciens carrying pBICI. Ginseng cells were then rinsed with sterilized water added 500 mg/L cefotaxime to kill the Agrobacterium tumefaciens on the surface of cells, and blotted dry on a sterilized paper towel, and placed onto a 67-V medium added 500 mg/L cefotaxime for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 500mg/L cefotaxime and 50mg/L G418 to select transgenic progeny. About 3～4 months later, G418-resistant ginseng cells regenerants were removed to a 67-V medium containing 25 mg/L G418. We have obtained the cell line of ginseng which carrying CTB-hIFN-α2b fusion gene.
Transgenic cells were checked for the presence of target gene using PCR and RT-PCR. Samples containing the target gene showed a clear band in site of 880 bp by agarose electrophoresis analysis. The results demonstrated that an amplified product with expected size was present in G418 resistant cells and absent in non-transformed cells. Expression products were carried out Western blot, anti-viral activity and GM1 binding assay analysis, the results showed that the expression protein of a human interferon and CTB protein properties biological activity.
From these results, it was concluded that fusion proteins can be expressed in transgenic ginseng callus cells and are able to keep the activity.
在本研究中,我们构建了含有霍乱毒素B亚单位与人干扰素α2b融合基因片段的植物细胞表达载体,选择人参愈伤组织细胞作为生物反应器,利用农杆菌介导的转化法将融合基因导入人参愈伤组织细胞,得到了能够同时表达hIFN-α2b和CTB的转基因人参愈伤组织细胞系。为建立以人参愈伤组织细胞作为生物反应器生产口服药用蛋白奠定了基础,并为药用蛋白和药用植物的联合应用开辟了一条新思路。
Human interferonα2b(hIFN-α2b)processes a variety of biological functions, including broad-spectrum antiviral, anti-cell division and immunomodulatory activities. In the clinic, it′s mainly for the treatment of viral hepatitis and cancer diseases. At present, the clinical application of interferon preparations are mainly obtained from the E.coli expression system, and the main administration is injection. Because the half-life is short, interferon would soon disappeare from the blood after the intravenous injection. It is difficult to maintain a high plasma concentration in clinical applications. The problem can only be solved through increasing the frequency and dosage. So that it is easy to produce interferon-induced antibody and increase the toxic side effects. The injected administration is inconvenient and expensive for patients. Therefore, it was need to develop new formulations of interferon.
Cholera toxin B subunit (CTB) is a non-toxic binding subunit of cholera toxin, it can specific bind with the gangliosides GM1 receptor which placed on the membrane of nucleated cells. CTB can be used for carrier protein, so exogenous peptides may be combined with CTB in order to increase the opportunity to contact with the cell membrane receptor. Based on these characteristics of CTB, today it has been applied to the oral vaccines and oral drugs research.
In recent years, the use of transgenic plants producing pharmaceutical proteins are able to in-depth study. Compared with the traditional system, the plant expression system is efficient, economical and convenient and so on. In addition, the most important edible plants also have characteristics. Therefore, the use of transgenic plants as bioreactors production of oral pharmaceutical proteins are being increasingly subject to national attention. It was report that plants can express high levels of functional recombinant proteins.
For transgenic plant material can be divided into plants, explant and callus cells, and so on. Plant tissue cell culture studies have decades of history. Compared with conventional agricultural production, it is not impacted by the ecological environment and the pests. Ginseng is a precious medicinal plants, has a unique nutritional and medicinal value. The common receptors of transgenic plants are tobacco, rice, tomato, potato and so on. The expression of human interferon in ginseng has not been reported.
In this study, human interferonα2b and cholera toxin B subunit gene was fused by gene fusion, which CTB was located at the N-terminal and hIFN-α2b was placed at the C-terminal. They were linked by a flexible peptide chain. First, the fusion gene was constructed into the pMD18-T vector to form the pMDCI. The pMDCI and pBI121 was digested with BamH I and Sac I. The target gene fragment and vector fragments were recovered by agarose gel electrophoresis and connected to receive expression vector. The recombinant plasmid was confirmed by PCR and restriction digestion, finally obtaining the plant expression vector pBICI.
The pBICI was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBICI was used to transform ginseng cells. Ginseng cells were co-cultivated for 48～72 h with Agrobacterium tumefaciens carrying pBICI. Ginseng cells were then rinsed with sterilized water added 500 mg/L cefotaxime to kill the Agrobacterium tumefaciens on the surface of cells, and blotted dry on a sterilized paper towel, and placed onto a 67-V medium added 500 mg/L cefotaxime for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 500mg/L cefotaxime and 50mg/L G418 to select transgenic progeny. About 3～4 months later, G418-resistant ginseng cells regenerants were removed to a 67-V medium containing 25 mg/L G418. We have obtained the cell line of ginseng which carrying CTB-hIFN-α2b fusion gene.
Transgenic cells were checked for the presence of target gene using PCR and RT-PCR. Samples containing the target gene showed a clear band in site of 880 bp by agarose electrophoresis analysis. The results demonstrated that an amplified product with expected size was present in G418 resistant cells and absent in non-transformed cells. Expression products were carried out Western blot, anti-viral activity and GM1 binding assay analysis, the results showed that the expression protein of a human interferon and CTB protein properties biological activity.
From these results, it was concluded that fusion proteins can be expressed in transgenic ginseng callus cells and are able to keep the activity.
引文
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