Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.)

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus ‘s’ gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.     

Hepatitis B surface antigen expression in NT-1 cells of tobacco using different expression cassettes

Nicotiana tabacum 1 (NT-1) cells were transformed with four different expression cassettes of hepatitis B surface antigen (HBsAg). The transformed nature of the cells was confirmed by polymerase chain reaction (PCR). The expression levels were assayed by enzyme linked immunosorbent assay (ELISA). The expressivities varied among the different cassettes and the maximum expression of 16.6 ng g⁻¹(f.m.) of cells was noted in pEFEHER transformed cells. Salicylic acid (100 μM) treatment resulted in 1.8 fold increase of expression in pEFEHBS transformed cells. The effect of different concentrations of kanamycin and geneticin was studied on the growth of transformed cells and HBsAg expression. The cell growth was optimum at lower concentrations of the antibiotics, and the maximum expression was noted at 200 mg dm⁻³ of kanamycin.     

Sucrose-inducible expression of hepatitis B surface antigen using potato granule-bound starch synthase promoter

NT-1 cells of tobacco (Nicotiana tabacum L.) were transformed with pGBSSHBS and pGBSSHER expression cassettes wherein expression of hepatitis B surface antigen (HBsAg) was driven by potato granule-bound starch synthase (GBSS) promoter. The transformed nature of the cells was confirmed by PCR analysis. Expression of HBsAg was confirmed by RT-PCR and Western blotting and levels of expression were assayed by ELISA. Transformed cell lines exhibited a sucrose-inducible pattern of HBsAg expression. NT-1 medium supplemented with 175 mmol L⁻¹ sucrose gave the highest HBsAg expression of 198 ng g⁻¹ FW after 8 days of induction. Different sugars, for example glucose, fructose, and palatinose, were also tested to study the inducible nature of GBSS promoter. The results demonstrate that potato GBSS promoter can be used in heterologous host systems like tobacco NT-1 cell suspension cultures for sucrose-inducible expression of recombinant proteins.     

Expression of hepatitis B small surface antigen in <Emphasis Type="Italic">Santalum album</Emphasis> embryogenic cell suspension cultures

Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated from the colony with expression of 11.09 μg(HBsAg) g⁻¹(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed the expression of 19.95 μg(HBsAg) g⁻¹(f.m.).     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.     

Organ-specific expression of hepatitis B surface antigen in potato

Summary The gene encoding the hepatitis B surface antigen (HBsAg) under the control of cauliflower mosaic virus 35S-promoter was constructed and expressed in transgenic potato plants. The HBsAg expression were measured by ELISA kit containing monoclonal antibodies. The amount of HBsAg in roots was found 5–10 fold higher than in leaf tissues     

Endosperm-specific expression of tyramine <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyltransferase and tyrosine decarboxylase from a single self-processing polypeptide produces high levels of tyramine derivatives in rice seeds

The plant-specific tyramine derivatives, feruloyltyramine (FT) and 4-coumaroyltyramine (CT), represent bioactive compounds found at low levels in many plant species. We generated transgenic rice seeds that produce high levels of CT (14 μg g⁻¹ seeds) and FT (2.7 μg g⁻¹ seeds) through the dual expression of tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase, using the self-processing foot-and-mouth disease virus 2A sequence and the endosperm-specific prolamin promoter.     

Generation of transgenic plants expressing plasma membrane-bound antibodies to the environmental pollutant microcystin-LR

In this paper we describe the engineering and regeneration of transgenic tobacco plants expressing a recombinant plasma membrane-retained antibody specific to microcystin-LR (MC-LR), the environmental toxin pollutant produced by cyanobacteria. The antibody was created by a genetic fusion of the antigen binding regions of the microcystin-specific single chain antibody, 3A8, with the constant regions from the murine IgG1κ, Guy’s 13, including a membrane retention sequence at the C-terminal end of the antibody heavy chain. The antibody produced in the leaves was shown to be functional by binding to MC-LR in an ELISA with antibody yields in transgenic plant leaves reaching a maximum of 1.2 μg g⁻¹ leaf f.wt (0.005% total soluble protein). Antibody-MC-LR complexes formed in leaves after addition of MC-LR to hydroponic medium around the roots of transgenic plant cultures.     

A new antibody immobilization strategy based on electro-deposition of gold nanoparticles and Prussian Blue for label-free amperometric immunosensor

A novel and sensitive immunosensor has been developed by electro-depositing gold nanoparticles on to a Prussian Blue-modified glassy carbon electrode for determination of hepatitis B surface antigen (HBsAg). After the developed immunosensor was incubated with different concentrations of HBsAg samples at 37°C for 15 min, the current response decreased with an increasing HBsAg concentration in the sample solution. The decreased percentage of the current was proportional to HBsAg concentration ranging from 2 to 300 ng HbsAg ml⁻¹ with a detection limit of 0.42 ng HbsAg ml⁻¹ (S/N = 3). Analytical results of 50 specimens using the developed immunosensor showed satisfactory agreement with those using ELISA, indicating the method to be a promising alternative for detecting HBsAg in clinical diagnosis.     

Identification and Characterization of the Glucosinolate–Myrosinase System in Caper (<Emphasis Type="Italic">Capparis ovata</Emphasis> Desf.)

Myrosinase (EC 3.2.1.147) catalyzes cleavage of glucosinolates, which consist of a thioglucoside moiety linked to amino acid-derived side chains. Myrosinase activity and expression profiles were investigated together with glucosinolate contents in Capparis ovata (caper) in order to characterize the glucosinolate–myrosinase system. The desulfoglucosinolates—glucocapparin, glucoiberin, progoitrin, epiprogoitrin, sinigrin, gluconapin, glucosinalbin, and glucobrassicin—were extracted and quantified from leaves, seeds, flowers, flower buds, and young shoots. The major desulfoglucosinolate was glucocapparin, which accumulated to values of 39.35 ± 0.09 and 25.56 ± 0.11 μmol g⁻¹ dry weight in seed and leaf extracts, respectively. Myrosinase has high activity in caper seeds, leaves, flowers, and flower bud tissues having the highest total activities in seed extracts (79.23 ± 0.18 U). However, specific activities were the highest in flower bud extracts (200.44 ± 0.09 U mg⁻¹ protein). The myrosinase protein migrated as a single band with a molecular weight of 65 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and on Western blots probed with the myrosinase-specific 3D7 antibodies. Native gel electrophoresis revealed two putative myrosinase isoenzymes in seeds, leaves, and flower tissues. The caper homolog of the Arabidopsis thaliana TGG1 gene was differentially expressed in seeds, leaves, flowers, and flower buds with the highest expression levels in leaves and flower bud tissues.     

Chemical-induced autoexcision of selectable markers in elite tomato plants transformed with a gene conferring resistance to lepidopteran insects

Marker-free transgenic tomato plants harboring a synthetic Bacillus thuringiensis endotoxin gene, cryIAc, were obtained by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination system, in which the selectable marker neomycin phosphotransferase gene flanked by two directly oriented loxP sites was located between the cauliflower mosaic virus 35S promoter and a promoterless cryIAc. Upon induction by 2 μM β-estradiol, sequences encoding the selectable marker and cre sandwiched by two loxP sites were excised from the tomato genome, leading to activation of the downstream endotoxin gene cryIAc with high expression levels as shown by Northern blot and ELISA assay (250–790 ng g⁻¹ fresh wt) in T₁ generation. For transgenic line with single transgenic loci, 15% of T₁ progenies were revealed marker-free. This autoexcision strategy provides an effective approach to eliminate a selectable marker gene from transgenic tomato, thus expediting the public acceptance of genetically modified crop.     

Functional analysis of an <Emphasis Type="Italic">iaaM</Emphasis> gene in parthenocarpic fruit development in transgenic <Emphasis Type="Italic">Physalis pubescens</Emphasis> L. plants

An efficient somatic embryogenesis system for Physalis pubescens L. (husk tomato) was developed prior to transformation. Subsequently, cotyledonary explants of P. pubescens were transformed with a chimeric construct containing an iaaM gene from driven by the fruit-specific promoter 2A12 to develop parthenocarpic fruits. Following selection of explants on Murashige and Skoog (MS) medium containing containing 75 mg l⁻¹ kanamycin (Km), 36 km-resistant callus clusters were recovered, and these were regenerated into whole plants. Expression of the iaaM gene was detected in confirmed transgenic fruits. The 0.9-kb 2A12 promoter was capable of directing expression of the introduced iaaM gene in transgenic P. pubescens fruits, but iaaM expression was absent from both leaves and flowers. Quantitative measurements of indole-3-acetic acid (IAA) content during fruit development indicated that the IAA levels in transgenic lines increased from anthesis through young fruits and peaked at fruit maturity. On average, IAA contents in transgenic fruits were two-fold higher than those in control fruits. Under greenhouse condition, vegetative growth, morphology, and the flowering of transgenic plants were comparable to those of control plants. However, the fruits of transgenic lines ripened earlier and had fewer seeds per fruit than did control plants.     

High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 µg/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.     

Transient expression of human papillomavirus type 16 virus-like particles in tobacco and tomato using a tobacco rattle virus expression vector

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g⁻¹(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.     

Effect of aminoglycoside antibiotics on in-vitro morphogenesis from cultured cells of chrysanthemum and tobacco

Successful genetic transformation of plants requires non-chimeric selection of transformed tissues and their subsequent regeneration. With rare exceptions, most transformation protocols still rely heavily on antibiotics for selecting transgenic cells that contain an antibiotic-degrading selectable marker gene. Here, the morphogenic capacity of in-vitro expiants of chrysanthemum and tobacco stems and leaves (control and transgenic) changed with the addition of aminoglycoside antibiotics (AAs). In a test of 6 AAs, phytotoxicity occurred at concentrations of 10 to 25 and 50 to 100 ng ml.⁻¹ in chrysanthemum and tobacco expiants, respectively. Light conditions as well as expiant source and size also had significant effects. The use of transverse thin cell layers (tTCLs), in conjunction with high initial AA selection levels, supported the greatest regeneration of transgenic material (adventitious shoots or callus) and the lowest number of escapes. Flow-cytometric analyses revealed no endoduplication in chrysanthemum, even at high AA levels. However, this phenomenon was observed in tobacco calli (8C or more), even at low AA concentrations (i.e., 5 to 10 μg mL^-1).     

乙型肝炎表面抗原基因转化花生及其表达

构建了乙肝表面抗原主蛋白基因(SHBs)的植物表达载体, 通过农杆菌介导转化花生(Arachis hypogaea)并利用潮霉素筛选出抗性苗, 经PCR和Southern杂交鉴定转基因植株; 取植株的蛋白粗提液进行ELISA检测, 结果表明, SHBs能在花生中表达, 且具有免疫原性, 其在新鲜叶片中的表达量约为2.41 mg.g-1鲜重, 占总可溶性蛋白的0.033%。     

Lead induced antioxidant defense system in pigeon pea and its impact on yield and quality of seeds

Toxic effects of lead (>0.2 mM Pb) were measured in pigeon pea (Cajanus cajan Mill) cv. UPAS grown in sand culture as reduction in growth, yield, and quality of seeds. Leaves containing >38 μg g⁻¹ Pb showed oxidative damage as decrease in chlorophyll content and induction of antioxidants such as carotenoids, proline, and non-protein thiol contents with enhanced activities of SOD and peroxidase. At excess (>0.2 mM) Pb, accumulation of >1,000 μg Pb g⁻¹ root tissue was associated with increase in non-protein thiol content. It is concluded that inhibition in root-to-shoot translocation of Pb and induction in the level of proline, chloroplast pigments, and non-protein thiols and activities of antioxidant enzymes SOD and peroxidase at <0.2 mM Pb could have protected the pigeon pea plants from the deleterious effects of Pb. However, excess Pb at >0.2 mM showed a decline in yield, boldness, and quality of seeds despite the expression of an additional band each of Cu–Zn SOD and peroxidase isoform. The threshold of toxicity and toxicity values in pigeon pea leaves of plants exhibiting 10 and 33% yield depression at 27 days after metal supply were 27 and 56 μg Pb g⁻¹, respectively.