人参愈伤组织细胞表达HBsAg-rhIFNα-2b融合蛋白的研究
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摘要
乙肝病毒表面抗原(HBsAg)基因已相继在大肠杆菌、酵母、哺乳动物细胞和昆虫杆状病毒体系中表达。其中产业化的有酵母和CHO系统两种。由于这两种系统表达的HBsAg制备的疫苗仍然有5-10%的人群不产生抗体,因此,人们一直在探索能够更有效刺激机体产生HBsAg抗体的新型疫苗。重组人干扰素α-2b(rhIFNα-2b)是治疗乙肝最有效的药物,在人参细胞中表达其与HBsAg的融和蛋白做为抗原,借助人参皂甙的免疫增强作用,有可能使机体产生更强的细胞免疫和体液免疫,并获得治疗性乙肝疫苗。
本研究构建了携带HBsAg-rhIFNα-2b融合基因的植物细胞表达载体,采用农杆菌感染方法将其整合到人参愈伤组织细胞的染色体上,得到了能够表达HBsAg-IFNα-2b融合蛋白的人参愈伤组织细胞系(150)。在试验中还建立了固体和液体培养两种培养系统。该细胞系不仅可以表达HBsAg-IFNα-2b融合蛋白,而且可以产生具有增强疫苗免疫效果作用的人参皂甙。本研究获得的基因工程乙肝疫苗无需纯化,也不用额外添加疫苗佐剂,直接免疫小鼠和家兔就能够产生有效的免疫应答。
转基因植物组织细胞作为生物反应器具有广阔的应用前景。本研究为利用植物组织培养技术表达和生产医用重组蛋白提供了技术平台,为其它珍稀药用植物的开发利用、组织培养及表达重组蛋白奠定了坚实基础。
Hepatits B virus infection is one of the most widespread viral infections of humans, especially in China. A highly effective and safe hepatitis B vaccine was licensed in the U.S. in 1986. It has a great commercial success, with sales in the industrialized world exceeding $ 1 billion every year. Why have we pursued to an alternative vaccine against hepatitis B virus (HBV) infections? The answer lies in finding an equivalent or improved vaccine to serve for developing countries of the world, where the HBV disease burden remains at epidemic levels, and the price of vaccine has precluded its popularity.
The existing vaccine against HBV infection is a biotechnological product that falls in the category of“subunit vaccines”. The gene coding the hepatitis B surface antigen (HBsAg) was expressed in yeast or CHO cells. The resulting vaccine is the epitome of modern macromolecular pharmaceuticals derived from recombinant DNA technology; it is a superior product that contains only a“subunit”of HBV. However, it is technology-intensive and expensive that the vaccine could not be a health-care product for developing countries.
Interferon is the most efficient drug for HBV, HBsAg-IFNα-2b fusion protein maybe get therapeutic vaccine. to aim directly at HBV.
In this paper, immunogenicities of recombinant hepatitis B surface antigens derived from CHO cells and in transgenic ginseng cells were compared. Transgenic cells material containing HBsAg was propitious to induce an immune response.
HBsAg-S gene was obtained from C28 cell containing the HBsAg-S DNA fragment using PCR and inserted into vector pMD18-T, to form pMDHS. IFN-α-2b gene was obtained from pBV888/DH5αcontaining the IFN-α-2b DNA fragment using PCR and inserted into vector pMD18-T to form pMDIF. The plasmid pMDHS and pGEM wer digested with BamHI and EcoRI, the DNA fragment recovered by electrophoresis was ligated into pGEHS. The plasmid pMDIF and pGEHS wer digested with EcoRI and SacI, the DNA fragment recovered by electrophoresis was ligated into clone vector pGESI. The plasmid pGESI and the binary plant vector pBI121 were digested by BamHI/SstI, the DNA fragment recovered by electrophoresis was ligated into pression vector pBISI.
The pBISI was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBISI was used to transform ginseng cells. Ginseng cells were co-cultivated for 48-72 h with Agrobacterium tumefaciens carrying pBISI. Ginseng cells were then rinsed with sterilized water added 500mg/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 500mg/L cefotaxime for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 300mg/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 35mg/L G418. We have obtained the cell line of ginseng, named 150 line which carrying HBsAg-IFNα-2b fusion gene. At the same time, 150 cell suspension culture system was constructed.
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 1300bp 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.
150 cells and negative control non-transformed ginseng cells were harvested, frozen in liquid N2, and ground to fine powder. The powder was suspended in extraction buffer (50mM Tris-HCl, 0.029% NaN3, PH9.5) at room temperature overnight. The surry was then centrifuged at 12000 rpm for 10 min at 4℃, and supernatants were tested using ELISA. Extracts of the 150 line expressed HBsAg detected by ELISA. Expression levels of HBsAg showed 224ng/g fresh weight, and the HBsAg amount was 0.009% of the total soluble protein. The untransformed ginseng cells did not express any protein that was reactive to antibody against HBsAg. HBsAg was expressed in intracellular and secreted forms in suspension culture 150 cells. Western blotting analysis confirmed the presence of 44 kDa band specific to HBsAg in 150 cells.
Forty-eight mice in six groups were injected. Group 1 were celiacly injected with 1 ml supernatants of 150 cells grounded. Group 2 were celiacly injected with 1 ml supernatants of C69 cells grounded and . a subimmunogenic dose (0.5ug HBsAg) of commercial HBV vaccine. Group 3 were celiacly injected with 1 ml supernatants of untransformed ginseng cells grounded. Group 4 were celiacly injected with a subimmunogenic dose (0.5ug HBsAg) of commercial HBV vaccine. Group 5 were celiacly injected without Al(OH)3 adjuvant. Group 6 were celiacly injected with 1ml normal saline. HBsAb in sera from either experimental or control animals was detected by ELISA, serum antibody of five mice was positive in group 1, serum antibody of four mice was positive in group 2,serum antibody of three mice was positive in group 4, serum antibody of all mice was negative in group 3, 5, and 6.
In the experiment of oral immunization, three experimental rabbits were fed with 80g 150 cells every ten days. Three control rabbits were fed with 80g untransformed ginseng cells. Serum antibody of two experimental rabbits were positive using ELISA, and serum antibody of all control rabbits were negative.
We have shown the expression of HBsAg in transgenic ginseng cells, and demonstrated that the plant-derived antigen was immunogenic to mice administered parenterally and to rabbits orally.
Over the past decade, increasing endeavors have been directed towards how to augment expression levels of foreign genes in transgenic plant cells. Some modifications of expression vectors have been successfully assayed to have the function to lead to improve levels of expression of the recombinant genes, including the use of stronger promoters, plant-optimized synthetic genes, plant-derived leader sequences, signal peptides which can target the protein for retention in intercellular compartments etc.
We have demonstrated that plant-derived rHBsAg was active as an immunogen. Transgenic Ginseng cells could induce specific immune response depending on the route of administration and immune status of the animal. In conclusion, more and more attentions have been paid to the study on vaccine using transgenic plants in the recent years, and many great achievements have been attained in this field. However, many problems still need to be solved. There is a long way to go before practical use of plant-derived vaccine.
本研究构建了携带HBsAg-rhIFNα-2b融合基因的植物细胞表达载体,采用农杆菌感染方法将其整合到人参愈伤组织细胞的染色体上,得到了能够表达HBsAg-IFNα-2b融合蛋白的人参愈伤组织细胞系(150)。在试验中还建立了固体和液体培养两种培养系统。该细胞系不仅可以表达HBsAg-IFNα-2b融合蛋白,而且可以产生具有增强疫苗免疫效果作用的人参皂甙。本研究获得的基因工程乙肝疫苗无需纯化,也不用额外添加疫苗佐剂,直接免疫小鼠和家兔就能够产生有效的免疫应答。
转基因植物组织细胞作为生物反应器具有广阔的应用前景。本研究为利用植物组织培养技术表达和生产医用重组蛋白提供了技术平台,为其它珍稀药用植物的开发利用、组织培养及表达重组蛋白奠定了坚实基础。
Hepatits B virus infection is one of the most widespread viral infections of humans, especially in China. A highly effective and safe hepatitis B vaccine was licensed in the U.S. in 1986. It has a great commercial success, with sales in the industrialized world exceeding $ 1 billion every year. Why have we pursued to an alternative vaccine against hepatitis B virus (HBV) infections? The answer lies in finding an equivalent or improved vaccine to serve for developing countries of the world, where the HBV disease burden remains at epidemic levels, and the price of vaccine has precluded its popularity.
The existing vaccine against HBV infection is a biotechnological product that falls in the category of“subunit vaccines”. The gene coding the hepatitis B surface antigen (HBsAg) was expressed in yeast or CHO cells. The resulting vaccine is the epitome of modern macromolecular pharmaceuticals derived from recombinant DNA technology; it is a superior product that contains only a“subunit”of HBV. However, it is technology-intensive and expensive that the vaccine could not be a health-care product for developing countries.
Interferon is the most efficient drug for HBV, HBsAg-IFNα-2b fusion protein maybe get therapeutic vaccine. to aim directly at HBV.
In this paper, immunogenicities of recombinant hepatitis B surface antigens derived from CHO cells and in transgenic ginseng cells were compared. Transgenic cells material containing HBsAg was propitious to induce an immune response.
HBsAg-S gene was obtained from C28 cell containing the HBsAg-S DNA fragment using PCR and inserted into vector pMD18-T, to form pMDHS. IFN-α-2b gene was obtained from pBV888/DH5αcontaining the IFN-α-2b DNA fragment using PCR and inserted into vector pMD18-T to form pMDIF. The plasmid pMDHS and pGEM wer digested with BamHI and EcoRI, the DNA fragment recovered by electrophoresis was ligated into pGEHS. The plasmid pMDIF and pGEHS wer digested with EcoRI and SacI, the DNA fragment recovered by electrophoresis was ligated into clone vector pGESI. The plasmid pGESI and the binary plant vector pBI121 were digested by BamHI/SstI, the DNA fragment recovered by electrophoresis was ligated into pression vector pBISI.
The pBISI was transformed into Agrobacterium tumefaciens stain LBA4404 directly by the freeze-thaw method. Subsequently, Agrobacterium tumefaciens carrying pBISI was used to transform ginseng cells. Ginseng cells were co-cultivated for 48-72 h with Agrobacterium tumefaciens carrying pBISI. Ginseng cells were then rinsed with sterilized water added 500mg/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 500mg/L cefotaxime for recovery. After recovery period of 7 days, the ginseng cells were transferred to a 67-V medium added 300mg/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 35mg/L G418. We have obtained the cell line of ginseng, named 150 line which carrying HBsAg-IFNα-2b fusion gene. At the same time, 150 cell suspension culture system was constructed.
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 1300bp 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.
150 cells and negative control non-transformed ginseng cells were harvested, frozen in liquid N2, and ground to fine powder. The powder was suspended in extraction buffer (50mM Tris-HCl, 0.029% NaN3, PH9.5) at room temperature overnight. The surry was then centrifuged at 12000 rpm for 10 min at 4℃, and supernatants were tested using ELISA. Extracts of the 150 line expressed HBsAg detected by ELISA. Expression levels of HBsAg showed 224ng/g fresh weight, and the HBsAg amount was 0.009% of the total soluble protein. The untransformed ginseng cells did not express any protein that was reactive to antibody against HBsAg. HBsAg was expressed in intracellular and secreted forms in suspension culture 150 cells. Western blotting analysis confirmed the presence of 44 kDa band specific to HBsAg in 150 cells.
Forty-eight mice in six groups were injected. Group 1 were celiacly injected with 1 ml supernatants of 150 cells grounded. Group 2 were celiacly injected with 1 ml supernatants of C69 cells grounded and . a subimmunogenic dose (0.5ug HBsAg) of commercial HBV vaccine. Group 3 were celiacly injected with 1 ml supernatants of untransformed ginseng cells grounded. Group 4 were celiacly injected with a subimmunogenic dose (0.5ug HBsAg) of commercial HBV vaccine. Group 5 were celiacly injected without Al(OH)3 adjuvant. Group 6 were celiacly injected with 1ml normal saline. HBsAb in sera from either experimental or control animals was detected by ELISA, serum antibody of five mice was positive in group 1, serum antibody of four mice was positive in group 2,serum antibody of three mice was positive in group 4, serum antibody of all mice was negative in group 3, 5, and 6.
In the experiment of oral immunization, three experimental rabbits were fed with 80g 150 cells every ten days. Three control rabbits were fed with 80g untransformed ginseng cells. Serum antibody of two experimental rabbits were positive using ELISA, and serum antibody of all control rabbits were negative.
We have shown the expression of HBsAg in transgenic ginseng cells, and demonstrated that the plant-derived antigen was immunogenic to mice administered parenterally and to rabbits orally.
Over the past decade, increasing endeavors have been directed towards how to augment expression levels of foreign genes in transgenic plant cells. Some modifications of expression vectors have been successfully assayed to have the function to lead to improve levels of expression of the recombinant genes, including the use of stronger promoters, plant-optimized synthetic genes, plant-derived leader sequences, signal peptides which can target the protein for retention in intercellular compartments etc.
We have demonstrated that plant-derived rHBsAg was active as an immunogen. Transgenic Ginseng cells could induce specific immune response depending on the route of administration and immune status of the animal. In conclusion, more and more attentions have been paid to the study on vaccine using transgenic plants in the recent years, and many great achievements have been attained in this field. However, many problems still need to be solved. There is a long way to go before practical use of plant-derived vaccine.
引文
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