Production and secretion of human interleukin-18 in transgenic tobacco cell suspension culture

Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18) was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under the control of a duplicated CaMV 355 promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using theAgrobacterium-mediated transformation method. The integration of the hIL-18 gene into the genome of transgenic tobacco plants was confirmed by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived from the transgenic plant callus by using Northern blot analysis. The hIL-18 protein was detected in the extracts of the transgenic callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked immunosorbant assay (ELISA) results, the expression level of the hIL-18 protein approximated 166 μg/L in the suspension culture medium. Bioassay results from the induction of interferon-γ from a KG-1 cell line indicated that the hIL-18 secreted into the suspension culture medium was bioactive.     

Expression of human growth hormone in transgenic rice cell suspension culture

Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D (Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension culture medium, and manifested biological activity.     

Co-expression of proteinase inhibitor enhances recombinant human granulocyte-macrophage colony stimulating factor production in transgenic rice cell suspension culture

The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production.     

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

启动子Ptac与PsbA在鱼腥藻7120中表达hG-CSF的效率比较

为了比较外源性启动子Ptac与内源性启动子PsbA在鱼腥藻7120中表达外源基因时的效率,构建了分别含Ptac和PsbA两种启动子的穿梭表达载体pRL-PsbA-GCSF、pRL-Tac-GCSF;利用三亲结合转移法转化鱼腥藻7120,利用抗生素筛选,通过质粒提取和PCR方法鉴定,获得了分别由2种启动子驱动表达hG-CSF的转基因蓝藻,转基因藻中目的基因以质粒形式存在;利用半定量RT-PCR方法对2种转基因藻的hG-CSF转录水平进行比较,发现PsbA启动子驱动效率与Ptac启动子没有明显差异;利用ELISA方法比较hG-CSF蛋白表达量,发现PsbA启动的蓝藻中hG-CSF表达量是Ptac诱导条件下表达量的1.17倍。     

Expression of a human anti-rabies virus monoclonal antibody in tobacco cell culture

A Nicotiana tabacum cv. Xanthi cell culture was initiated from a transgenic plant expressing a human anti-rabies virus monoclonal antibody. Within 3 months, plant cell suspension cultures were established and recombinant protein expression was examined. The antibody was stably produced during culture growth. ELISA, protein G purification, Western blotting, and neutralization assay confirmed that the antibody was fully processed, with association of light and heavy-chains, and that it was able to bind and neutralize rabies virus. Quantification of antibody production in plant cell suspension culture revealed 30 microg/g of cell dry weight for the highest-producing culture (0.5 mg/L), 3 times higher than from the original transgenic plant. The same production level was observed 3 months after cell culture initiation. Plant cell suspension cultures were successfully grown in a new disposable plastic bioreactor, with a growth rate and production level similar to that of cultures in Erlenmeyer flasks.     

Secretory production of hGM-CSF with a high specific biological activity by transgenic plant cell suspension culture

The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about 180 μg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to 270 μg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than that of a commercially available preparation fromE. coli.     

A 3,387?bp 5′-flanking sequence of the goat alpha-S1-casein gene provides correct tissue-specific expression of human granulocyte colony-stimulating factor (hG-CSF) in the mammary gland of transgenic mice

A new expression vector containing the 1,944 bp 5'-flanking regulatory region together with exon 1 and intron 1 of the goat alpha-S1-casein gene (CSN1S1), the full-sized human granulocyte colony-stimulating factor gene (hGCSF) and the 3'-flanking sequence of the bovine CSN1S1, was created. The vector DNA was used for generation of four mouse transgenic lines. The transgene was integrated into chromosomes 8 and 12 of two founders as 2 and 5 copies, respectively. Tissue-specific secretion of hG-CSF into the milk of transgenic mice was in the range of 19-40 μg/ml. RT-PCR analysis of various tissues of the transgenic mice demonstrated that expression of hGCSF was detected in only the mammary gland in the progeny of all founders. Moreover, cells were shown to be positive for hG-CSF by immunofluorescent analysis in the mammary glands but not in any other tissues. There were no signs of mosaic expression in the mammary gland. Trace amounts of hG-CSF were detected in the serum of females of two transgenic lines during lactation only. However, no transgenic mice showed any changes in hematopoiesis based on the number of granulocytes in blood. Immunoblotting of hG-CSF in the milk of transgenic mice revealed two forms, presumably the glycosylated and non-glycosylated forms. The hematopoietic activity of hG-CSF in the milk of transgenic females is comparable to that of recombinant G-CSF. In general, the data obtained in this study show that the new expression vector is able to provide correct tissue-specific expression of hG-CSF with high biological activity in transgenic mice.     

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.     

新城疫病毒融合蛋白在转基因水稻叶片中的表达及其免疫原性

利用转基因植物作为生物反应器表达抗原蛋白具有广阔的应用前景。以新城疫病毒融合蛋白（NDVF）基因1.7kb全长编码区序列为外源基因与组成型表达的玉米泛素蛋白基因（Ubi）启动子和农杆菌胭脂碱合成酶基因（nos）终止子组成嵌合基因,构建了适宜于农杆菌介导转化水稻的转化载体pUNDV,经根癌农杆菌介导的遗传转化方法将由Ubi动子驱动的NDVF嵌合基因导入水稻细胞中,经潮霉素抗性筛选,共再生获得了6个独立的转基因株系。PCR分析结果表明NDVF基因已整合到水稻基因组中。ELISA和Western blot分析结果证实NDVF蛋白在部分转基因水稻叶片组织中获得表达,其中植株F5叶片组织中具有较高的表达水平。将F5叶片可溶性总蛋白皮下注射免疫BALB／c小鼠,结果表明能够诱导小鼠产生一定水平的NDVF蛋白特异抗体。     

Biological activity of human granulocyte colony stimulating factor with a modified C-terminus

Granulocyte colony-stimulating factor (G-CSF) undergoes receptor-mediated internalization into target cells which are normally restricted to neutrophilic granulocytes and their committed progenitor cells, suggesting that it may be applicable as a myeloid cell-targeting vehicle. To test this notion, we constructed a cDNA encoding a human G-CSF/murine stem cell factor (mSCF) chimeric molecule in a mammalian expression vector and transfected NIH3T3 cells with this plasmid. The resulting chimeric cytokine consisted of the entire G-CSF sequences fused to Lys148 of mSCF. It can be released from the surface membrane of NIH3T3 transformants through proteolytic cleavage at Ala164 of mSCF. The culture media conditioned by a number of stable transformants, which were confirmed by an enzyme-linked immunosorbent assay (ELISA) to secrete an hG-CSF derivative, were examined for their ability to stimulate CFU-G-derived colony formation as well as the proliferation of G-CSF-dependent NFS-60 cells. The results indicated that this C-terminus modified version of hG-CSF is as potent as recombinant hG-CSF in both assays.     

Inducible expression of Norwalk virus capsid protein gene in plant cell suspension cultures

Plant cell suspension cultures can be used to make safe vaccines at a lower cost than conventional procedures. An inducible gene expression system provides an opportunity to optimize the conditions of vaccine production in a plant system. In this investigation, a dexamethasone-inducible Norwalk virus capsid protein (NVCP) gene expression system has been developed in cell suspension cultures for four different plant species: tobacco (Nicotiana tabacum), rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.) via Agrobacterium-mediated transformation. Resulting transgenic cell lines were confirmed by Southern blot analyses and NVCP gene expression was confirmed by Northern blot analysis. NVCP gene expression was observed in all 24 cell lines tested, but there were minor differences in transgene expression among the transgenic cell lines. The highest level of NVCP gene expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone (10 mg/l), for all transgenic cell lines derived from four different plant species. This investigation demonstrated that expression of NVCP in different transgenic cell lines and in different species was tightly controlled by the inducer, and the inducible gene expression system could be useful in controlling expression of NVCP or similar proteins for production of vaccines in cultured plant cells.     

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Expression of Rice Stripe Virus Coat Protein Gene in Transgenic Rice Plants

Rice stripe virus (RSV) is a pathogen of rice stripe disease causing great damage to rice. The disease is transmitted by Laodelphax striatellus and three other planthoppers. RSV infects as much as 37 cereals including rice, wheat, maize and results in a significant reduction in yield in epidemic year. In order to develop efficient means of controlling the disease, authors have studied the amino acid composition of RSV coat protein (CP), synthesized and cloned the cDNA to CP, sequenced the full-length CP gene. Having inserted the RSV CP gene into plant expression vector pROK Ⅱ, authors transformed rice suspension culture via microprojectile bombardment and obtained transgenic plants expressing the CP gene. The suspension culture was initiated by inoculating yellowish, compact and embryogenic calli derived from seeds into suspension medium containing proline and maltose. After being cultured at 26℃ in the dark for about half a year, finely-dispersed and embryogenic suspension culture was estabolished. Before bombardment the suspension culture was evently applied onto three-layered filter-paper discs in a petri dish. CaCl2 and spermidine was employed to coat tungsten particle with plasmid DNA. 2.5 μl of coated particle was loaded onto bullet and each dish was bombarded three times. Immediately after being bombarded, the suspensions were cultured in modified N6 medium. 2 days later the suspensions were transferred to the same medium but containing G418, which were subcultured weekly. Being subject to G418 selection for two months, white and fast-growing clones were emerged from the brownish cultures. Green plants regenerated when the resistant calli were transferred to differentiation medium. The regenerated plants were firm enough to grow well in the greenhouse. 10 plants regenerated from G418 resistant calli were tested for their transformed nature by Southern blot using 32P-labelled CP gene as a probe. Among the plants tested, 2 plants showed clearly hy bridizing bands with a molecular weight corresponding to RSV CP gene. Western blot further demonstrated that RSV CP gene was expressed in transgenic rice plants. At present tests on the antiviral effects of transgenic plants by feeding plantphoppers infccted with RSV are being underway.     

Tumor targeting of humanized fragment antibody secreted from transgenic rice cell suspension culture

The tumor-associated glycoprotein 72 (TAG 72) has been shown to be expressed in the majority of human adenocarcinomas. In an effort to develop a technique for the safe and inexpensive production of large quantities of anti-TAG 72 humanized antibody fragments (hzAb) as a future source of clinical-grade proteins, we developed a transgenic rice cell suspension culture system. The in vivo assembly and secretion of hzAb were achieved in a transgenic rice cell culture under the control of the rice alpha amylase 3D (RAmy 3D) expression system, and the biological activities of plant-derived hzAb were determined to be quite similar to those of animal-derived antibody. Purified hzAb was shown to bind to the recombinant antigen, TAG 72, and to bind specifically to human LS 174T colon adenocarcinoma cells expressing the TAG 72 antigen, and this binding occurred to the same extent as was seen with animal-derived antibody. Plant-derived hzAb proved as effective as animal-derived antibody in targeting tumors of xenotransplanted LS 174T cells in nude mice. The results of this study indicate that the hzAb derived from plant cell suspension cultures may have great potential for pharmaceutical applications in the development of future cancer therapeutic and diagnostic protocols.