Xiaolin Meng

PcLT, a novel C-type lectin from Procambarus clarkii, is involved in the innate defense against Vibrio alginolyticus and WSSV

Lectins play important roles in the innate immunity. In this work, a C-type lectin, PcLT, was obtained from Procambarus clarkii which contained a carbohydrate recognition domain (CRD) with the ability to bind to Vibrio alginolyticus and white spot syndrome virus (WSSV). RT-PCR and qRT-PCR analyses demonstrated PcLT was specifically expressed in the hepatopancreas and the mRNA was markedly upregulated by V. alginolyticus and WSSV challenge, although a slight difference in timing was observed. The study also revealed upregulation of the mRNA expression and activity of immunological factors, peroxinectin, phenoloxidase, and superoxide dismutase in hemolymph in response to recombinant PcLT (rPcLT). Moreover, rPcLT also enhanced the phagocytosis, facilitated the subsequent clearance of V. alginolyticus and prolonged the survival of WSSV-infected shrimp. These results suggested that PcLT not only served as a pathogen recognition receptor (PRR), but also functioned as an immune modulator, participating in host defense against invaders.

Development of Lateral-flow Immunoassay for WSSV with Polyclonal Antibodies Raised against Recombinant VP (19+28) Fusion Protein

We have developed a sensitive and rapid lateral-flow immunoassay (LFIA) for WSSV, using colloidal gold as an indicator. The fusion protein, VP (19+28), was expressed in E. coli, purified and used to prepare polyclonal antibodies. The purified anti-VP (19+28) IgG were conjugated with colloidal gold. Unconjugated anti-VP (19+28) IgG and goat anti-rabbit IgG were immobilized on nitrocellulose membranes. After assembly, three groups (5 individual animals in each group) of shrimp samples were tested which included healthy, moribund and dead shrimps. For each group, three different tissues (body juices, gills and hepatopancreas) were tested at the same time. In parallel, all the samples were also analyzed using PCR for comparison. Out of 45 samples tested, 30 were detected as positive while 15 were classified as negative. The results of LFIA correlate with those obtained by the PCR analysis, indicating that these two detection methods have the same efficacy in the limited number of samples tested in this preliminary study.

Improve bioavailability of Harpin protein on plant use PLGA based nanoparticle

Harpins can induce systemic acquired resistance (SAR) pathway on scores of non-host plant, provide protection against a range of pathogens. In this study, we demonstrated that applied recombinant HarpinZ Pseudomonas syringae pv. tomato (rHrpZ) on tobacco with three kinds of methods: infiltrating from micro-pore into leaf; injecting into petiole, and spraying on leaf, there is great difference in assimilation of protein because of the poor osmosis of tobacco leaves, and with multi-application of rHrpZ, the stimulation effect decreased. We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ PLGA NPs). To study the drug effect, we analyzed the change ratio of phenylalanine ammonia lyase (PAL) activity and PR-5dB gene expression after administration of rHrpZ and rHrpZ PLGA NPs on tobacco leaves. The results show that rHrpZ could elicit a rapid and transient increase in both PAL activity and PR-5dB expression, but the effect decreased after multi-application. While sprayed rHrpZ PLGA NPs on leaves, both the change ratio of PAL activity and PR-5dB expression were comparatively smooth and durable. Our study suggested that rHrpZ NPs could help protein enter leaf epidermis and cell wall, release rHrpZ in situ continuously, and enhance the bioavailability of rHrpZ.

Antitumor activity of recombinant antimicrobial peptide penaeidin-2 against kidney cancer cells

SummaryPenaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

The fusion protein Penhrp containing Penaeidin-2 and HarpinZ induced a stronger hypersensitive response and systemic acquired resistance in tobacco comparing to the original HarpinZ

In the present study, the gene (pen2) encoding a mature Penaeidin-2 from Penaeus vannamei. shrimp and gene (rHarpinZ) encoding a HarpinZ from Pseudomonas syringae pv. tomato were respectively cloned. The pen2 was fused to rHarpinZ with a linker (GGSGSGGSSRGGSGS) and then cloned into pET-32a (+) formed the expression plasmid pET-32a (+)/pen-hrp. Penhrp was expressed in the prokaryotic (E. coli Origami (DE3) pLysS) expression system. Functional analysis showed that the fusion protein Penhrp could elicit stronger hypersensitive response (HR) including higher Peroxidase (POD) and Superoxide dismutase (SOD) activities and the transcription of PR-5dB gene than the original HarpinZ. In addition, a stronger systemic acquired resistance (SAR) to TMV in Xanthi nc. tobacco was also induced. These may offer a potential effective strategy for plant disease control.