Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview

During the past two decades, antibodies, antibody derivatives and vaccines have been developed for therapeutic and diagnostic applications in human and veterinary medicine. Numerous species of dicot and monocot plants have been genetically modified to produce antibodies or vaccines, and a number of diverse transformation methods and strategies to enhance the accumulation of the pharmaceutical proteins are now available. Veterinary applications are the specific focus of this article, in particular for pathogenic viruses, bacteria and eukaryotic parasites. We focus on the advantages and remaining challenges of plant-based therapeutic proteins for veterinary applications with emphasis on expression platforms, technologies and economic considerations.     

Hydrolysis of organophosphate compounds by mutant butyrylcholinesterase: a story of two histidines

This study is aimed at understanding the hydrolysis mechanism of organophosphate (OP) compounds by G117H-BChE. It is a theoretical study that focuses on the role of the G117H mutation in the dephosphorylation step. Various proposed mechanisms are examined. We show that His117 acts as a general base by activating a water molecule, and thus assisting its nucleophilic attack on the phosphate. The calculated reaction energy profile agrees well with the experimental data. Moreover, analysis of the reaction via its two hypothetical elementary steps, proton transfer and hydroxide attack, supports the role of His117 as a general base. Further support to the proposed mechanism is gained by structural comparison of the active site to RNAse A, which has similar composition of substrate and functional groups. The similarity between these enzymes extends beyond the structure and also becomes evident when comparing functionality of various active sites residues as well as rate-pH dependence obtained in the two cases. Moreover, it is demonstrated that an extended form of Bevilacqua's model (Biochemistry 2003;42:2259-2265) may resolve the apparent contradictions between the proposed mechanism and various experimental observations regarding rate-pH dependence. Finally, that same model is shown to rationalize the hydrolase activity of G117D BChE, an observation which is considered puzzling. It is concluded that G117H-BChE hydrolyzes echothiophate and possibly other OP compounds via a general acid-base mechanism. On the basis of this mechanism, one can now proceed with rational design aimed at improving the enzyme by exploiting both the structural and mechanistic knowledge.     

Intra- and extra-cellular organophosphorus hydrolase production with recombinant <Emphasis Type="Italic">E. coli</Emphasis> using fed-batch fermentation

A fed-batch fermentation process for the production of organophosphorus hydrolase (OPH) (EC 3.1.8.1) by E. coli pET812 was developed in this research. With batch fermentation, the maximum OPH concentrations attained by batch fermentation were as low as 4 × 10⁵ U/l because cell growth and OPH production were inhibited by a high initial concentration of glucose. To develop a fed-batch fermentation process for obtaining higher concentrations of OPH, highly concentrated glucose solution (500 g/l) was added intermittently or continuously to increase the carbon source concentration. Eventually, 3.2 × 10⁶ U/l of OPH was produced with fed-batch fermentation in 24 h. This was eight times higher than the yield with conventional batch fermentation. A total concentration of 399–441 mg of OPH was produced/l, which was four times higher than that reported when using E. coli. Nearly half (44%) of the produced OPH was secreted into the culture solution.     

Transgenic plants as a source for the bioscavenging enzyme,human butyrylcholinesterase

Organophosphorous pesticides and nerve agents inhibit the enzyme acetylcholinesterase at neuronal synapses and in neuromuscular junctions. The resulting accumulation of acetylcholine overwhelms regulatory mechanisms, potentially leading to seizures and death from respiratory collapse. While current therapies are only capable of reducing mortality, elevation of the serum levels of the related enzyme butyrylcholinesterase (BChE) by application of the purified protein as a bioscavenger of organophosphorous compounds is effective in preventing all symptoms associated with poisoning by these toxins. However, BChE therapy requires large quantities of enzyme that can easily overwhelm current sources. Here, we report genetic optimization, cloning and high‐level expression of human BChE in plants. Plant‐derived BChE is shown to be biochemically similar to human plasma‐derived BChE in terms of catalytic activity and inhibitor binding. We further demonstrate the ability of the plant‐derived bioscavenger to protect animals against an organophosphorous pesticide challenge.     

Genetically engineered resistance to organophosphate herbicides provides a new scoreable and selectable marker system for transgenic plants

Organophosphate hydrolase (OPH, E.C. 3.1.8.1; encoded by the bacterial opd gene) provides a new scoreable and selectable genetic marker system for use in plant cell culture and regenerated plant tissue. OPH hydrolyzes a wide range of substrates that produce visually detectable products, which can be readily quantified in biological tissues. A variety of different OP compounds, both herbicides and pesticides, have been identified as acceptable enzymatic substrates, which can be used to generate transgenic markers for various types of plant tissues. For example, transgenic leaf tissue was easily differentiated from non-transgenic tissue by a simple fluorescent assay utilizing the OP insecticide coroxon. Transformed callus and intact whole seed could be easily distinguished from non-transformed tissue using novel non-destructive methods which allowed callus or seeds to grow and/or to germinate after phenotypic scoring with non-herbicidal OP insecticides such as paraoxon. In addition to being used as a scoreable phenotypic markers with various OP pesticides, the OP compounds Haloxon and Bensulide (Bensumec-4LF) were effective as positive selection agents for callus and germinating seeds.     

分子医药农业研究进展

分子医药农业是利用转基因植物为载体,以农业生产的方式规模化生产各种有治疗用途的重组蛋白质及多肽。近20年来,随着植物生物反应器技术的多元化发展以及日趋成熟,植物分子医药农业产业悄然而生。近几年,一些分子医药农业生产的植物源医药产品已实现规模化生产,并进入市场。文中结合国内外最新的研究进展,重点对几种主要植物生物反应器的研究、产品的规模化生产以及产业化进程进行了阐述,以期为我国分子医药农业领域的研究与应用提供参考。     

High-value products from transgenic maize

Maize (also known as corn) is a domesticated cereal grain that has been grown as food and animal feed for tens of thousands of years. It is currently the most widely grown crop in the world, and is used not only for food/feed but also to produce ethanol, industrial starches and oils. Maize is now at the beginning of a new agricultural revolution, where the grains are used as factories to synthesize high-value molecules. In this article we look at the diversity of high-value products from maize, recent technological advances in the field and the emerging regulatory framework that governs how transgenic maize plants and their products are grown, used and traded.     

Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin

Reverse peptide of indolicidin (Rev4), a 13-residue peptide based on the sequence of indolicidin, has been shown to possess both strong antimicrobial and protease inhibitory activities in vitro. To evaluate its efficacy in vivo, we produced and evaluated transgenic tobacco (Nicotiana tabacum L.) and Arabidopsis thaliana [(L.) Heynh.] plants expressing Rev4 with different signal peptide sequences for pathogen resistance. All transgenic plants showed normal growth and development, an indication of no or low cytotoxicity of the peptide. Furthermore, the transgenic plants exhibited elevated resistance to three bacterial and two oomycete pathogens. Interestingly, tobacco plants expressing Rev4 displayed enhanced yield compared to the control as indicated by an increased biomass production by as much as 34% in two field trials. When Rev4 was coexpressed with another antimicrobial peptide, Myp30, the disease resistance levels in the transgenic Arabidopsis were enhanced. These findings suggest the potential of using these peptides to protect plants from microbial pathogens and to enhance yield.     

新型白化型除草剂靶标酶对羟苯基丙酮酸双加氧酶及其耐性转基因植物研究进展*

对羟苯基丙酮酸双加氧酶(ρ-hydroxyphenylpyruvate dioxygenase,HPPD;EC 1.13.11.27)催化生物体内对羟苯基丙酮酸与O2作用形成尿黑酸的反应,是植物体中质体醌和生育酚生物合成途径的关键酶。当其活性受到抑制时,植物体中作为类胡萝卜素生物合成途径中最终电子受体和光合链电子传递体的质体醌的生物合成受阻,进而导致类胡萝卜素合成减少,光合链电子传递受阻,致使植物体出现白化症状。目前已经开发了多种以HPPD为靶标的除草剂,该类除草剂及抗除草剂转基因植物研究具有广阔的前景。对这一新型白化型除草剂靶标酶以及耐该类除草剂转基因植物的研究进展作了简要综述。     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.     

Highly selective carbamate-based butyrylcholinesterase inhibitors derived from a naturally occurring pyranoisoflavone

This current study described the design and synthesis of a series of derivatives based on a natural pyranoisaflavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26 compounds showed significant inhibition on BChE (the IC₅₀ values varied from 9.34 μM to 0.093 μM), most of them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds was performed. Among them, 9g (IC₅₀ = 222 nM) and 9h (IC₅₀ = 93 nM) were found to be the most potent BChE inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a good candidate of BChE inhibitors.     

高效删除标记基因的Bhlea2植物表达载体的构建

为培育去除选择标记基因的耐旱转基因植物,同时利用Cre/Lox和FLP/frt系统,构建一个能够高效删除标记基因的Bhlea2基因植物表达载体.拟南芥rd29A启动子是在低温、干旱、高盐胁迫下的快速应答启动子,玉米ubiquitin启动子可有效驱动外源基因的转录,拟南芥pAB5启动子是花粉及胚胎等发育早期特异表达的启动子,利用上述启动子构建了表达Bhlea2基因并能够删除标记基因的植物表达载体.该表达载体包括重组酶表达元件pAB5-FLP、Bhlea2抗旱基因表达元件rd29A-Bhlea2和bar标记基因表达元件ubiquitin-bar.     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

植物生物反应器优化策略与最新应用*

随着分子生物学和植物基因工程的迅猛发展以及分子医药和现代农业等学科的交叉融合,植物生物反应器已成为分子医药农业的核心内容。利用植物生物反应器生产抗体、疫苗和功能性食品,具有规模化、成本低、安全性高、周期短等优势。2022年2月,加拿大卫生部批准了新型冠状病毒疫苗Covifenz^®,这是世界首款植物源人体疫苗,标志着以植物生物反应器为代表的分子医药农业时代的来临。综述植物叶片和种子等代表性的植物生物反应器类型,分析瞬时表达系统和稳定表达系统的构建原理和应用,探讨通过启动子和密码子优化、糖基化过程“人源化”、基因沉默抑制和蛋白酶作用抑制等优化植物生物反应器的策略,总结国内外抗体、疫苗和功能性食品等植物源产品的开发进展,以期为我国植物生物反应器的研究及其在分子医药领域的应用提供参考。     

Oxidation at C-16 enhances butyrylcholinesterase inhibition in lupane triterpenoids

A set of triterpenoids with different grades of oxidation in the lupane skeleton were prepared and evaluated as cholinesterase inhibitors. Allylic oxidation with selenium oxide and Jones’s oxidation were employed to obtain mono-, di- and tri-oxolupanes, starting from calenduladiol (1) and lupeol (3). All the derivatives showed a selective inhibition of butyrylcholinesterase over acetylcholinesterase (BChE vs. AChE). A kinetic study proved that compounds 2 and 9, the more potent inhibitors of the series, act as competitive inhibitors. Molecular modeling was used to understand their interaction with BChE, the role of carbonyl at C-16 and the selectivity towards this enzyme over AChE. These results indicate that oxidation at C-16 of the lupane skeleton is a key transformation in order to improve the cholinesterase inhibition of these compounds.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Paraoxonase (PON 1) as a biomarker of susceptibility for organophosphate toxicity

Paraoxonase (PON1) is an A-esterase capable of hydrolysing the active metabolites (oxons) of a number of organophosphorus (OP) insecticides such as parathion, diazinon and chlorpyrifos. PON1 activity is highest in liver and plasma, and among animal species significant differences exist, with birds and rabbits displaying very low and high activity, respectively. Human PON1 has two polymorphisms in the coding region (Q192R and L55M) and five polymorphisms in the promoter region. The Q192R polymorphism imparts different catalytic activity toward some OP substrates, while the polymorphism at position -108 (C/T) is the major contributor to differences in the level of PON1 expression. Animal studies have shown that PON1 is an important determinant of OP toxicity, with animal species with a low PON1 activity having an increased sensitivity to OPs. Administration of exogenous PON1 to rats or mice protects them from the toxicity of OPs. PON1 knockout mice display a high sensitivity to the toxicity of diazoxon and chlorpyrifos oxon, but not paraoxon. In vitro assayed catalytic efficiencies of purified PON₁₉₂ isoforms for hydrolysis of specific oxon substrates accurately predict the degree of in vivo protection afforded by each isoform. Low PON1 activity may also contribute to the higher sensitivity of newborns to OP toxicity.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411

Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus (OP) agent at alkaline pH, digested with pepsin at pH 2.3, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Two singly charged peaks m/z 1718 and 1831, corresponding to the unlabeled peptide fragments containing the active site Tyr411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr411. The binding of DFP to Tyr411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr411. The presence of Tyr411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.