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.     

Expression and purification of functional human alpha-1-Antitrypsin from cultured plant cells

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.     

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.     

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.     

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.     

Production of bioactive human granulocyte-colony stimulating factor in transgenic rice cell suspension cultures

Human granulocyte-colony stimulating factor (hG-CSF), a human cytokine, was expressed in transgenic rice cell suspension culture. The hG-CSF gene was cloned into the rice expression vector containing the promoter, signal peptide, and terminator derived from a rice alpha-amylase gene Amy3D. Using particle bombardment-mediated transformation, hG-CSF gene was introduced into the calli of rice (Oryza sativa) cultivar Dong-jin. Expression of the hG-CSF gene was confirmed by ELISA and Northern blot analysis. The amount of recombinant hG-CSF accumulated in culture medium from transgenic rice cell suspension culture on the sugar starvation was determined by time series ELISA. Biological activity of the plant derived hG-CSF was confirmed by measuring the proliferation of the AML-193 cells, and was similar to that of the commercial Escherichia coli-derived hG-CSF. In this paper, we discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant hG-CSF.     

Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice Proteinase Inhibitor

With the primers designed basing on the terminal amino acid sequences of rice proteinase inhibitors and the preferred codons of rice genes, a new gene coding for a rice proteinase inhibitor has been amplified and cloned from Oryza sativa var. japonica (cv. Zhonghua 8) using PCR technique. The gene contains 408 basepairs and encodes 133 amino acid residues. The deduced amino acid sequence with duplicated Bowman-Birk type structure and active sites specific to trypsin has relatively high homology with that of proteinase inhibitors from wheats, beans etc. As for rice, the new gene shares 74.8% homology with a rice bran trypsin inhibitor reported previously. The evolutionary characteristics of the proteinase inhibitor family has also been discussed.     

一种水稻蛋白酶抑制剂基因的克隆及其结构分析

参照水稻蛋白酶抑制剂部分氨基酸序列 ,利用水稻偏爱密码子设计引物 ,经 PCR扩增 ,从我国水稻 (Oryza sativa)品种“中花 8号”中克隆到一个长 40 8bp的基因。序列测定和分析表明 ,克隆到的是一个未见报道的新的水稻蛋白酶抑制剂基因 ,该基因编码了一个由 1 33个氨基酸组成 ,具有重复双功能结构域和以抑制胰蛋白酶为主的活性中心的包曼 -伯克 (Bowman- Birk)型蛋白酶抑制剂 ,该基因推导的氨基酸序列与大麦、小麦、豆类等的某些蛋白酶抑制剂的氨基酸序列具有较高的同源性 ,与该家族的水稻的一种胰蛋白酶抑制剂氨基酸全序列同源性高达 75%。     

水稻半胱氨酸蛋白酶抑制剂基因的克隆及其mRNA在水稻中的组成型表达

从40年代发现豆科植物中存在蛋白酶蛋白抑制剂以来,在动物、植物和微生物体内已发现普遍存在着多种类型的蛋白酶抑制剂(PI)。人们往往是为了研究某种蛋白酶的作用机制或出于某种应用目的去分离和研究PI的,对PI的真正生理功能尚不十分清楚。一般认为除防止体内不必要的蛋白降解作用、调节蛋白代谢及调节各种蛋白酶的生理活性外,很多植物的PI还具有抑制某些病源微生物及某些昆虫体内蛋白酶的作用,从而对植物有防卫功能。Hilder等和Johnson等已分别将属于丝氨酸蛋白酶抑制剂的豇豆蛋白酶抑制剂及马铃薯PⅠⅠ和PⅠⅡ基因转入烟草,结果转基因烟草对烟芽夜蛾(He-     

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.     

Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.     

Production of human serum albumin by sugar starvation induced promoter and rice cell culture

Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice α Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the α Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2–4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the α Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.     

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.     

不同启动子驱动下马铃薯蛋白酶抑制剂PinⅡ转基因水稻的遗传、表达和对粘虫抗性分析

以不同启动子驱动的马铃薯蛋白酶抑制剂Ⅱ基因(PinⅡ-2x ,PinⅡ-4x)转基因水稻为材料,经潮霉素抗性、PCR和Southern blot等检测对转基因水稻后代进行了遗传分析。结果显示：外源基因在68.4%的转基因植株中符合孟德尔遗传模式,单拷贝植株率为63.6%。转基因植株后代的PinⅡ蛋白活性测定结果表明：ActI和Ubi驱动的PinⅡ-2x转基因水稻植株中,每克鲜叶片的PinⅡ蛋白含量为160μg和176μg,而由PIN5′驱动的PinⅡ-4x为104μg,对照水稻仅为20μg。ActI和Ubi驱动的PinⅡ表达产物对胰蛋白酶活性抑制程度分别达到37.7%和43.1%,明显高于PinⅡ自身启动子PIN5′（29.2%）。叶片饲养粘虫幼虫的实验表明：转基因植株叶片对粘虫有抗性,但抗性达不到显著水平,且启动子效率、PinⅡ表达量与抗粘虫      

水稻悬浮细胞再生及根癌农杆菌介导转化的影响因素

已经成功报道的农杆菌介导的水稻遗传转化多以活力较高的胚性愈伤为材料,很少以水稻悬浮细胞作为受体．另外,利用农杆菌转化多数都是通过浸泡的方式进行侵染．本实验利用滴加浸染法进行农杆菌介导转化水稻悬浮细胞,探讨影响 DNA 转化效率的因素．研究显示,在转化前,将水稻悬浮细胞在愈伤诱导培养基上培养1～2周,诱导产生直径为2～3 mm的微小愈伤组织对转化非常重要.微小愈伤组织大小不应小于 2 mm;对悬浮细胞短时间培养不但会缩短植株再生时间,而且会提高转化效率．此外,侵染农杆菌的浓度、侵染时间和不同侵染方法也影响 T-DNA 插入基因组的效率．用 1 ml Ａ600值为 0.5 浓度的农杆菌悬液滴加在水稻悬浮细胞诱导的愈伤,培养3 d或直到可见农杆菌菌落,此方法可以得到较高转化效率．将再生的潮霉素抗性的转化植株在含有 50 mg/L 潮霉素的分化和生根培养基中筛选得到,并对转化植株 gus 基因的表达进行 PCR 检测.结果显示,用 Ａ600值为 0.5 浓度的农杆菌浸泡侵染 20 min和滴加浸染法,分别得到PCR阳性植株率为 70% 和92%.