Vipin Kumar Deo

Multiple co-transfection and co-expression of human β-1,3-N-acetylglucosaminyltransferase with human calreticulin chaperone cDNA in a single step in insect cells

Human β‐1,3‐N‐acetylglucosaminyltransferase 2 (β3GnT2) is indispensable for the conversion of lacto‐N‐triose II into lacto‐N‐tetraose and lacto‐N‐neotetraose. In this paper, we report multiple co‐transfection in a single step using two different human cDNAs in an insect cell, β3GnT2 and calreticulin chaperone respectively. This minimized the time required to isolate stably expressing cell line from 12 weeks to 4 weeks and simplified the isolation technique to a one‐step process. We tried to insert as much cDNA as possible and used various concentrations of two antibiotics, Blasticidin and Geneticin, at 25–1500 μg/ml respectively during co‐transfection for the selection of an efficiently expressing stable cell line with no adverse effects. A stably expressing cell line was isolated which expressed β3GnT2 and chaperone simultaneously, which gave an activity of 10.1 m‐units/ml compared with 6.7 m‐units/ml by a cell only carrying β3GnT2. In this study we correlated the activity of β3GnT2 with the amount of β3GnT2 and human calreticulin cDNA in a stably expressing insect cell line simultaneously expressing calreticulin chaperone. When the amounts of chaperone and β3GnT2 cDNA were in a rough ratio of 1:1, the β3GnT2 activity was at a high level. In order to achieve better expression levels of β3GnT2 with less cost and time, efficient ways have to be devised.

Expression of an RSV-gag virus-like particle in insect cell lines and silkworm larvae

Rous sarcoma virus group antigen protein-based virus-like particles (VLPs) are well known for their structural integrity and ease of handling. VLPs play an important role in drug delivery systems because they can be manipulated with ease. In this study, a new method was established for expressing Rous sarcoma virus group antigen protein based VLPs in silkworm larvae and establishing stably expressing insect cell lines. These VLPs have been isolated by ultracentrifugation using a sucrose step gradient of 10-60% (v/v), and their spherical structure has been confirmed using transmission electron microscopy (TEM). The spherical morphology is similar in both the silkworm larvae and in stably expressing cell lines. Silkworm larvae are better suited for producing Rous sarcoma virus group antigen protein-based VLPs on a large scale; yields from silkworm larvae were approximately 8.2-fold higher than yields from stable cell lines. These VLPs provide a new method for large-scale application in vaccine development and drug delivery systems.

Display of Neospora caninum surface protein related sequence 2 on Rous sarcoma virus-derived gag protein virus-like particles

Virus-like particles (VLPs) displaying antigen have been increasingly recognized as a potential vaccine in the livestock industry. In this study, Neospora caninum surface protein related sequence (NcSRS)2 was displayed on the surface of Rous sarcoma virus group-antigen protein (RSV-gag) VLPs. Two types of Bombyx mori nucleopolyhedrovirus (BmNPV) bacmids, encoding RSV-gag and NcSRS2 genes, were co-injected into silkworm larvae to produce VLPs-NcSRS2. At 7 days post-injection, VLPs-NcSRS2 were collected from hemolymph and purified. The antigenicity of the purified protein was confirmed by enzyme-linked immunosorbent assay (ELISA) using neosporosis-positive bovine serum. ELISA revealed that ~0.16μg rNcSRS2 was displayed per 1μg VLPs-NcSRS2. To develop an antibody specific for VLPs-NcSRS2, purified VLPs-NcSRS2 were used to immunize mice in a three-dose regimen without adjuvant and the production of antibodies was confirmed in serum samples. By using a silkworm expression system, we demonstrated the display, expression and immunization of neosporosis-targeting membrane proteins, which are vaccine candidates for neosporosis.

Production of Rous sarcoma virus-like particles displaying human transmembrane protein in silkworm larvae and its application to ligand–receptor binding assay

Two types of Rous sarcoma virus (RSV) group-antigen protein (Gag) virus like particles (VLPs), full-length Gag (Gag701) and RSV protease domain (PR)-deleted mutant (Gag577) were expressed in silkworm larvae. Gag577 was secreted into hemolymph efficiently using wild type bacmid (WT), cysteine protease-deficient bacmid (CP(-)), cysteine protease and chitinase-deficient bacmid (CP(-)Chi(-)) bacmids, but comparatively Gag701 secretion levels were low. VLPs were purified on 10-60% (v/v) sucrose density gradient by ultracentrifugation and their structures confirmed under electron microscope. When hPRR and RSV Gag577 were co-expressed in silkworm larvae, human prorenin receptor (hPRR) was displayed on the surface of RSV VLPs, which was detected by Western blotting and immunoelectron microscopy. Moreover, binding of hPRR localized on the surface of VLPs to human prorenin was confirmed by ELISA. These results indicate that active hPRR was displayed on the surface of RSV VLPs, which can be utilized for drug discovery of hPRR blockers to prevent nephropathy. Moreover, this transmembrane protein display system using RSV Gag in silkworm larvae is applicable to expression of intact transmembrane proteins and binding assay of transmembrane proteins to its ligands, especially for transmembrane proteins which cannot be purified from membrane fractions in active states.

Chimeric Virus-Like Particles Made Using GAG and M1 Capsid Proteins Providing Dual Drug Delivery and Vaccination Platform

Nanobiomaterials such as enveloped virus-like particles with specificity can serve a dual function of vaccination and drug delivery system. Here, we expressed colon carcinoma cell-targeting chimeric virus-like particles (VLPs) made using two capsid proteins, gag and M1 from influenza virus A/swine flu/Iowa/15/30/H1N1 in silkworms. These chimeric VLPs displayed a glycosylphosphatidylinositol-anchored single-chain variable fragment region targeting colon carcinoma cells, and their shape was smooth, with an average particle size of 21 nm in diameter. Large unilamellar vesicles made from DOPC:DOPA (2:1) containing calcein-AM (10 μM) or doxorubicin (13.7 nM) were used to package chimeric VLPs. VLPs showed high specificity in targeting cancer cells and delivered the dye and drug to cells successfully. Chimeric VLPs were injected into BALB/c mice, and the serum showed specificity for M1 protein as a model.

Development of Rous sarcoma Virus-like Particles Displaying hCC49 scFv for Specific Targeted Drug Delivery to Human Colon Carcinoma Cells

PurposeVirus-like particles (VLPs) have been used as drug carriers for drug delivery systems. In this study, hCC49 single chain fragment variable (scFv)-displaying Rous sarcoma virus-like particles (RSV VLPs) were produced in silkworm larvae to be a specific carrier of an anti-cancer drug.MethodRSV VLPs displaying hCC49 scFv were created by the fusion of the transmembrane and cytoplasmic domains of hemagglutinin from influenza A (H1N1) virus and produced in silkworm larvae. The display of hCC49 scFv on the surface of RSV VLPs was confirmed by enzyme-linked immunosorbent assay using tumor-associated glycoprotein-72 (TAG-72), fluorescent microscopy, and immunoelectron microscopy. Fluorescein isothiocyanate (FITC) or doxorubicin (DOX) was incorporated into hCC49 scFv-displaying RSV VLPs by electroporation and specific targeting of these VLPs was investigated by fluorescent microscopy and cytotoxicity assay using LS174T cells.ResultsFITC was delivered to LS174T human colon adenocarcinoma cells by hCC49 scFv-displaying RSV VLPs, but not by RSV VLPs. This indicated that hCC49 scFv allowed FITC-loaded RSV VLPs to be delivered to LS174T cells. DOX, which is an anti-cancer drug with intrinsic red fluorescence, was also loaded into hCC49 scFv-displaying RSV VLPs by electroporation; the DOX-loaded hCC49 scFv-displaying RSV VLPs killed LS174T cells via the specific delivery of DOX that was mediated by hCC49 scFv. HEK293 cells were alive even though in the presence of DOX-loaded hCC49 scFv-displaying RSV VLPs.ConclusionThese results showed that hCC49 scFv-displaying RSV VLPs from silkworm larvae offered specific drug delivery to colon carcinoma cells in vitro. This scFv-displaying enveloped VLP system could be applied to drug and gene delivery to other target cells.

A new compound from the mushroomTricholoma flavovirens

A novel compound (1) and a known one (2) were isolated from the fruiting bodies of Tricholoma flavovirens. Their structures were determined by the interpretation of spectroscopic data. Both compounds showed inhibition effects on the growth of hypocotyl of lettuce with significant differences. In addition, compound 1 showed a promotion effect on the growth of root with significant differences and 2 had the similar tendency to promote the growth.

Virus-Like Particles Displaying Recombinant Short-Chain Fragment Region and Interleukin 2 for Targeting Colon Cancer Tumors and Attracting Macrophages

Functionalized virus-like particles (VLPs) can target with specificity as drug delivery systems and can attract macrophages for the destruction of cancer cells. Here, the group antigen capsid protein from the Rous sarcoma virus was used to prepare VLPs, functionalized by displaying glycol-inositol phosphate-anchored recombinant single chain fragment variable (rscFv) and hemagglutinin transmembrane region anchored recombinant human interleukin-2 (rhIL2) (designated as VLP-rscFv-rhIL2s) in silkworms. The rscFv specifically binds the tumor-associated glycoprotein 72 that is expressed at the surface of colon cancer cells. VLP-rscFv-rhIL2 was affinity purified and had a smooth particle size with a diameter of 50 nm. Calcein-AM-packaged VLP-rscFv-rhIL2s successfully targeted cancer cells as a model for drug delivery system. VLP-rscFv-rhIL2 bound with colon cancer cells that attracted macrophages (human monocytic cell line-1 cells) in chemotaxis chamber assays compared with negative controls. The macrophages secreted tumor necrosis factor-α, a cytokine that is necessary to destroy cancer cells. These results demonstrate the potential of VLP-rscFv-rhIL2 as an intelligent nano biomaterial that is capable of attracting macrophages.

Functional Virus-Like Particles Production Using Silkworm and Their Application in Life Science

Virus-like particles (VLPs), which mimic the structure of authentic virus, are of significant importance owing to their wide ranging applications. VLPs have the potential to serve as candidate vaccine in addition to subunit vaccines and also serve as a nano-bioparticle scaffold for displaying complex foreign proteins. Here, we review different methods available to produce VLPs with various host expression systems including from microorganisms to mammalian cells and the current potential of silkworms as producers of these VLPs. Recently, silkworms have been used for efficient production of VLPs too in addition to mass production of recombinant proteins, which have contributed to molecular structural analysis, molecule–molecule interactions in biology and biotechnology.

Quantitative screening of insect cell transformants stably expressing GFPuv-ß 1,3-N-acetylglucosaminyltransferase 2 fusion protein

Insect cell transformants, stably expressing human ß1,3-N-acetylglucosaminyltransferase 2 (ß3GnT2) as the green fluorescent protein (GFPuv)-fused protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and-pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ß3GnT activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.