Production of a functional single-chain Fv protein in transgenic tobacco root exudates

Nicotiana tabacum was transformed with a gene encoding anti-PreS1 of hepatitis B surface antigen single-chain Fv antibody (scFv) and bearing an N-terminal endoplasmic reticulum protein signal peptide sequence. The scFv antibody protein was continuously secreted from the transgenic tobacco roots into a simple hydroponic medium at 630 to 760 ng g^–1 dry wt root day^–1. The antibody was about 2% of the total secreted protein and still possessed antigen-binding activity.     

Expression and long-term stability of a recombinant single-chain Fv antibody fragment in transgenic Nicotiana tabacum seeds

A functionally active anti-hepatitis B surface antigen single-chain Fv antibody fragment (scFv) was expressed in seeds of transgenic tobacco plants using genetic constructs for expression in the vacuole or the apoplastic fluid. Antibody levels close to 0.2% of the total soluble protein were found. After storage of the transgenic tobacco seeds for one year and a half a year at room temperature, the scFv maintained its antigen-binding activity in full.     

Expression of a single-chain Fv antibody fragment specific for the Hepatitis B surface antigen in transgenic tobacco plants

An anti-Hepatitis B virus surface antigen (HBsAg) single chain Fv (scFv) antibody fragment was expressed in Nicotiana tabacum transgenic plants. The 6-histidine tagged scFv was targeted to either the cytosol, apoplast, and vacuole, or for retention in the endoplasmic reticulum. Expression of active scFv was detected by ELISA in fresh leaf material from F1 transgenic plant lines representative of the genetic constructs targeting the antibody fragment to the apoplastic fluid (AF-12, 0.031% of the total soluble protein), vacuole (V-20, 0.032% of the total soluble protein), and endoplasmic reticulum (ER-52, 0.22% of the total soluble protein). No scFv was detected by ELISA or western blot in the plants transformed with the cytosol construct. The biologically active scFv was easily purified (to 94–95% purity) from ER-52 and AF-12 plant material using immobilized metal ion affinity chromatography. Recovery estimated from the ER-52 plant line indicates that 15–20g of pure active scFv can be obtained per gram of fresh leaf material, on a laboratory scale.     

Expression of anti human IL-4 and IL-6 scFvs in transgenic tobacco plants

The two murine single-chain Fv (scFv) genes against human interleukin IL-4 and IL-6 cytokines were cloned in a plant expression vector (pGEJAE1) and mobilized to Agrobacterium tumefaciens. Tobacco leaf discs were co-cultured with Agrobacterium and transferred to selective media for regeneration. The tobacco in vitro plants produced scFvs against human IL-4 and IL-6. Only 8% of transformed plants expressing anti-IL-4 scFv were obtained versus 76% of transformed plants expressing anti-IL-6 scFv. In addition, some plants producing anti-IL-4 and anti-IL-6 scFvs aged more rapidly in in vitro conditions and in greenhouse pots than did control plants. Western blot analysis showed that the transformed Nicotiana tabacum plants contained proteins with an apparent molecular mass on electrophoresis of ca. 32 kDa, corresponding to the predicted size of the scFvs. As entire plant root seemed to accumulate more scFv than did leaves, we decided to continue working with isolated roots. Anti-IL-6 scFvs were detected in cultivated roots and their culture media. Functional anti-IL-6 scFv accounted for 0.16–0.18% of total soluble proteins. The affinity of the anti-IL-6 scFv produced in plants and measured by Biacore was similar to that of scFv produced in Escherichia coli. The high levels of antibody accumulation in isolated roots and secretion into the medium demonstrate the potential for producing recombinant protein in bioreactor systems.these authors contributed equally to this workthese authors contributed equally to this work     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Expression of a single-chain human interleukin-12 gene in transgenic tobacco plants and functional studies

Interleukin 12 (IL-12) is a key heterodimeric cytokine produced by a variety of antigen-presenting cells, including dendritic cells, macrophages, and B cells. It displays a potent array of biological activities affecting natural killer (NK) and T cells. These activities include promotion of cell-mediated or type 1 T helper cell responses (Th1). Due to that property, IL-12 has been employed in cancer immunotherapy, in mouse models of infectious diseases and in airway inflammation, and it may also have utility as a vaccine adjuvant. Transgenic plants are being used in many laboratories around the world for the production of therapeutically valuable proteins and as vehicles for oral vaccines. Here we present the expression of a single-chain human interleukin-12 in transgenic tobacco plants. The biological activity of plant-produced IL-12 was determined by interferon gamma (IFN-gamma) production by natural killer (NK) cells, and the level of production was comparable to that obtained with commercially available recombinant IL-12. The potential use of this recombinant protein is discussed.     

Combination of Dsb coexpression and an addition of sorbitol markedly enhanced soluble expression of single-chain Fv in Escherichia coli

Many eukaryotic proteins have been produced successfully in Escherichia coli. However, not every gene can be expressed efficiently in this organism. Most proteins, especially those with multiple disulfide bonds, have been shown to form insoluble protein or inclusion body in E. coli. An inactive form of protein would require an in vitro refolding step to regain biological functions. In this study, we described the system for soluble expression of a single-chain variable fragment (scFv) against hepatocellular carcinoma (Hep27scFv) by coexpressing Dsb protein and enhancing with medium additives. The results revealed that overexpression of DsbABCD protein showed marked effect on the soluble production of Hep27scFv, presumably facilitating correct folding. The optimal condition for soluble scFv expression could be obtained by adding 0.5M sorbitol to the culture medium. The competitive enzyme-linked immunosorbent assay (ELISA) indicated that soluble scFv expressed by our method retains binding activity toward the same epitope on a hepatocellular carcinoma cell line (HCC-S102) recognized by intact antibody (Ab) (Hep27 Mab). Here, we report an effective method for soluble expression of scFv in E. coli by the Dsb coexpression system with the addition of sorbitol medium additive. This method might be applicable for high-yield soluble expression of proteins with multiple disulfide bonds.     

A plant signal sequence enhances the secretion of bacterial ChiA in transgenic tobacco

When the secreted bacterial protein ChiA is expressed in transgenic tobacco, a fraction of the protein is glycosylated and secreted from the plant cells; however most of the protein remains inside the cells. We tested whether the efficiency of secretion could be improved by replacing the bacterial signal sequence with a plant signal sequence. We found the signal sequence and the first two amino acids of the PR1b protein attached to the ChiA mature protein directs complete glycosylation and secretion of the ChiA from plant cells. Glycosylation of this protein is not required for its efficient secretion from plant cells.     

Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy

Field pea (Pisum sativum L.) appears well suited for the production of high-value molecules such as recombinant antibodies, with well-established agricultural practices world-wide and seeds that are easily stored and distributed. In order to evaluate the suitability of this grain legume for the production of biologically active antibodies, we transformed peas with a cDNA encoding the single-chain Fv fragment scFvT84.66. This scFv is derived from the monoclonal antibody T84.66, which recognises the well-characterised tumour-associated carcinoembryonic antigen. The antibody is useful for in vitro immunodiagnosis and in vivo imaging of human cancers. We expressed scFvT84.66 cDNA under the control of the seed-specific legumin A promoter. We targeted the antibody to the endoplasmic reticulum for better stability and high accumulation. Transgenic plants produced up to 9 g per gram fresh weight of functional scFvT84.66 in their seeds. The transgene was stably inherited and expressed in the progeny, and the antibody remained active after storage in dried transgenic seeds for two months at room temperature. Our results demonstrate the suitability of grain legume seeds to produce biologically active recombinant antibodies, and the utility of field pea seeds as production vehicles for recombinant pharmaceutical macromolecules.     

Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants

The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model. An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2). After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration. Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring. The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb). By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell. Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb. The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb. Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Rapid Purification of a New Humanized Single-chain Fv Antibody／Human Interleukin-2 Fusion Protein Reactive against HER2 Receptor

Overexpression of the proto-oncogene c-erbB-2, neuor HER2 has been shown in many human tumor cells,especially in breast cancer cells [1–5], which is thought tobe important in human carcinogenesis. c-erbB-2 geneencodes a 185 kD transmembrane glycoprotein …     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T₁ progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T₁ progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T₁ plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T₁ progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.     

Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures

Six different expression cassettes of hepatitis B surface antigen (HBsAg) were used to transform tobacco cell suspension cultures. The transgenic nature of the cells was confirmed by PCR. The secreted HBsAg was assayed by ELISA and analyzed by Western blotting. A maximum of 31 μg antigen/l was obtained in the spent medium from the transformed cells. The use of an ethylene-forming enzyme promoter and incorporation of C-terminal endoplasmic-reticulum-retention signal enhanced the secretion of HBsAg. Salicylic or jasmonic acid at 10 μM increased secretion of HBsAg by six fold.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

转铁蛋白受体单链抗体与链亲和素融合基因的克隆及其在大肠杆菌中的可溶性表达

目的:转铁蛋白受体特异性富含于血脑屏障和肿瘤细胞的表面,是当前中枢神经系统疾病和肿瘤治疗中定向转运的重要靶标。拟获得在大肠杆菌中能高效可溶表达的转铁蛋白受体单链抗体与链亲和素(SA)的重组融合蛋白。方法:根据GenBank数据库报道的SA的核苷酸序列分段合成基因,连接后经PCR获得完整的基因片段,插入pGEM-T载体中测序。将序列正确的SA基因与大鼠转铁蛋白受体单链抗体基因ox26-scFv分别插入原核表达载体pTIG-Trx中,构建重组表达克隆pTIG-Trx/scFv-SA,并在大肠杆菌中诱导表达。ELISA检测融合蛋白的生物学活性。结果:对pGEM-T/SA克隆的测序结果显示,合成的SA基因与文献报道相符。重组融合蛋白在大肠杆菌中获得了可溶性表达,约占菌体上清总蛋白量的30%;ELISA结果表明该融合蛋白具备与转铁蛋白受体和生物素的结合的双重活性。结论:有活性的重组融合蛋白的获得为构建一个通用性的以转铁蛋白受体介导的血脑屏障和肿瘤转运靶向载体打下了基础。     

High expression of a human lactoferrin in transgenic tobacco cell cultures

Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.