Search Results (5)

Lysozyme is an enzyme that hydrolyzes bacterial cell walls, which is functional for destroying the integrity of bacteria, enhancing the activity of immune cells, participating in immune signal transmission, helping to maintain the micro-ecological balance of the gastrointestinal tract, etc. Egg white lysozyme (EWL), as one of the typical representatives of lysozyme, is the most widely used enzyme in production so far, and is also one of the most complex structures of lysozyme. EWL also helps protect plants from fungal and bacterial diseases. Here, we report the effect of EWL on infections from plant viruses. The EWL gene was cloned and characterized. The EWL protein sequence analysis identified a conserved domain of lysozyme activity and the sharing of a 100% identical EWL protein from the Coturnix japonica lysozyme. Then, the EWL gene was cloned into the plant expression vector pEAQ-HT-DEST3 and transiently expressed in Nicotiana benthamiana (N. benthamiana). We found that EWL expression in N. benthamiana significantly contributed to infections by the turnip mosaic virus (TuMV) but not by the tobacco mosaic virus (TMV). Plants that transiently expressed EWL showed an obvious increase in resistance to Botrytis cinerea (B.cinerea). Our results suggested a new research point for the application of EWL on plant pathogen infections. Full article

Isoflavonoids, the diverse group of secondary metabolites derived from the phenylpropanoid pathway, are distributed predominantly in leguminous plants. It has received considerable attention in recent days due to its health promoting benefits and is known to prevent certain diseases in humans. These isoflavonoids are synthesized from flavonoid intermediates of phenylpropanoid pathway by the enzyme isoflavone synthase. Metabolic engineering of isoflavonoid biosynthesis in non-legume crop plants could offer the health benefits of these compounds in diverse plant species further contributing for crop improvement. The transient expression of heterologous genes in the host is considered as an alternative to stable expression, that can provide a rapid way of studying the pathway engineering for metabolite production and could also act as a production platform for nutraceuticals and biopharmaceuticals. In this study, isoflavone genistein was produced in Amaranthus tricolor var. tristis and Spinacia oleracea by transiently expressing Glycine max isoflavone synthase (GmIFS). The GmIFS gene was cloned in plant expression vector pEarleyGate 102 HA and pEAQ-HT-DEST 3 and transformed into plants by agroinfiltration. The presence of transgene in the agroinfiltrated leaves was confirmed by semiquantitative reverse-transcription polymerase chain reaction. The flavonoid substrate naringenin and isoflavonoid genistein were quantified using high performance liquid chromatography in both wild-type and infiltrated leaf samples of both the plants. The naringenin content varied in the range of 65.5–338.5 nM/g fresh weight, while the accumulation of genistein was observed with varying concentrations from 113 to 182.6 nM/g fresh weight in the agroinfiltrated leaf samples of both A. tricolor var. tristis and S. oleracea. These results indicate that the transient expression of GmIFS gene has led to the synthesis of isoflavonoid genistein in A. tricolor var. tristis and S. oleracea providing an insight that stable expression of this gene could enrich the nutraceutical content in the crop plants. To the best of our knowledge, this is the first report on transient expression of GmIFS gene for the production of genistein in A. tricolor var. tristis and S. oleracea. Full article

The production of plant helical virus-like particles (VLPs) via plant-based expression has been problematic with previous studies suggesting that an RNA scaffold may be necessary for their efficient production. To examine this, we compared the accumulation of VLPs from two potexviruses, papaya mosaic virus and alternanthera mosaic virus (AltMV), when the coat proteins were expressed from a replicating potato virus X- based vector (pEff) and a non-replicating vector (pEAQ-HT). Significantly greater quantities of VLPs could be purified when pEff was used. The pEff system was also very efficient at producing VLPs of helical viruses from different virus families. Examination of the RNA content of AltMV and tobacco mosaic virus VLPs produced from pEff revealed the presence of vector-derived RNA sequences, suggesting that the replicating RNA acts as a scaffold for VLP assembly. Cryo-EM analysis of the AltMV VLPs showed they had a structure very similar to that of authentic potexvirus particles. Thus, we conclude that vectors generating replicating forms of RNA, such as pEff, are very efficient for producing helical VLPs. Full article

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine. Full article

Mistletoe (Viscum album) extracts have been used as alternative and complementary therapeutic preparations in multiple cancers for decades. Mistletoe lectins (ML-I, ML-II, and ML-III) are considered to be the main anticancer components of such preparations. In the present study, ML-II was transiently expressed in Nicotiana benthamiana using the pEAQ-HT expression system. Expression levels of up to 60 mg/kg of the infiltrated plant tissue were obtained, and a three-fold increase was achieved by adding the endoplasmic reticulum (ER) retention signal KDEL to the native ML-II sequence. The native protein containing His-tag and KDEL was purified by immobilized metal affinity chromatography (IMAC) and gel filtration. We found that the recombinant ML-II lectin was glycosylated and retained its carbohydrate-binding activity. In addition, we demonstrated that plant produced ML-II displayed anticancer activity in vitro, inhibiting non-small cell lung cancer H460 and A549 cells with EC50 values of 4 and 3.5 µg/mL, respectively. Annexin V-448A and PI double staining revealed that cell cytotoxicity occurred via apoptosis induction. These results indicate that ML-II transiently expressed in N. benthamiana plants is a promising candidate as an anticancer agent, although further optimization of production and purification methods is required to enable further in vitro testing, as well as in vivo assays. Full article