Sf9 Cell Membranes Research Articles

Development of Polycistronic Baculovirus Surface Display Vectors to Simultaneously Express Viral Proteins of Porcine Reproductive and Respiratory Syndrome and Analysis of Their Immunogenicity in Swine.

To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in E. coli. In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 105 of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines.

RNA interference of PHB1 enhances virulence of Vip3Aa to Sf9 cells and Spodoptera frugiperda larvae.

In our previous work, we demonstrated that prohibitin 2 (PHB2) on the membrane of Sf9 cells was a receptor for Vip3Aa, and PHB2 in mitochondria contributed to the mitochondrial stability to reduce Vip3Aa toxicity. Prohibitin 1 (PHB1), another prohibitin family member, forms heterodimers with PHB2 to maintain the structure and stability of mitochondria. To explore whether PHB1 impacts the action process of Vip3Aa, we examined the correlation between PHB1 and Vip3Aa virulence. We revealed that PHB1 did not colocalize with Vip3Aa in Sf9 cells. The pulldown and CoIP experiments confirmed that PHB1 interacted with neither Vip3Aa nor scavenger receptor-C (another Vip3Aa receptor). Downregulating phb1 expression in Sf9 cells did not affect the internalization of Vip3Aa but increased Vip3Aa toxicity. Further exploration revealed that the decrease of phb1 expression affected mitochondrial function, leading to increased ROS levels and mitochondrial membrane permeability and decreased mitochondrial membrane potential. The increase of mitochondrial cytochrome c release, caspase-3 activity and genomic DNA fragmentation implied that the apoptotic process was also affected. Finally, we applied RNAi to inhibit phb1 expression in Spodoptera frugiperda larvae. As a result, it significantly increased Vip3Aa virulence. We found that PHB1 was not a receptor for Vip3Aa but played an essential role in mitochondria. The downregulation of phb1 expression in Sf9 cells caused instability of mitochondria, and the cells were more prone to apoptosis when challenged with Vip3Aa. The combined use of Vip3Aa and phb1 RNAi showed a synergistic effect against S. frugiperda larvae. © 2023 Society of Chemical Industry.

A nano-delivery system expands the insecticidal target of thiamethoxam to include a devastating pest, the fall armyworm.

Nano-delivery systems have been applied to deliver various synthetic/botanical pesticides to increase the efficiency of pesticide use and reduce the volumes of pesticides applied. Previous studies have supported the hypothesis that the nanocarriers can help expand the insecticidal target of pesticides to include non-target pests. However, the potential mechanism underlying this interesting phenomenon remains unclear. Herein, a widely applied star polycation (SPc) nanocarrier was synthesized to construct a thiamethoxam (TMX) nano-delivery system. The SPc-based delivery system could promote the translocation of exogenous substances across the membrane of Sf9 cells, increase the cytotoxicity of TMX against Sf9 cells by nearly 20%, and expand the insecticidal target of TMX to include Spodoptera frugiperda (the fall armyworm), with a 27.5% mortality increase at a concentration of 0.25mg/mL. Moreover, the RNA-seq analysis demonstrated that the SPc could upregulate various transport-related genes, such as Rab, SORT1, CYTH, and PIKfyve, for the enhanced cellular uptake of TMX. Furthermore, enhanced cell death in larvae treated with the TMX-SPc complex was observed through changes in the expression levels of death-related genes, such as Casp7, BIRC5, MSK1, and PGAM5. The SPc-based nano-delivery system improved the cellular uptake of TMX and expanded its insecticidal target by adjusting the expression levels of death-related genes. The current study mainly identified the transport and cell death genes related to nanocarrier-based insecticidal target expansion, which is beneficial for understanding the bioactivity enhancement of the nano-delivery system.

A Tale of Two Scavenger Receptors: Structure‐Function Relationships of Purified SR‐B1 and CD36

Both CD36 and SR‐B1 are glycosylated integral membrane proteins belonging to the class B scavenger receptor family and are known to play roles in cardiovascular and atherosclerotic disease. CD36 serves as the receptor for oxidized low density lipoprotein (oxLDL) and facilitates cholesterol accumulation in macrophages and development of atherosclerotic plaques. SR‐B1 (scavenger receptor class B type 1) is the primary receptor for high density lipoprotein (HDL), which removes cholesterol from the peripheral atherosclerotic plaques and delivers it to the liver for excretion. Current structural information about class B scavenger receptors is limited to only purified extracellular regions, transmembrane segments, or transient transfection studies in cultured cells, and remains a roadblock to studying the isolated functions of these lipid and cholesterol transporters. Recently, we have successfully expressed and purified full‐length human SR‐B1 and CD36 using an Sf9 insect cell/baculoviral infection system, which allows for the expression of glycosylated proteins. Purified full‐length receptors are a valuable addition to our scientific toolbox and allow us to ask novel questions about the structure‐function relationships of scavenger receptors. To verify function within Sf9 cell membranes, we expressed human full‐length constructs in plated Sf9 cells and observed significantly increased binding of DiI‐HDL and DiI‐oxLDL, as well as increased DiI‐lipid uptake when comparing SR‐B1 and CD36 to empty vector infected cells, respectively. When purified and solubilized in detergent micelles, both receptors remain stable over time as shown by Prometheus thermal shift assays. We have also demonstrated that purified proteins are able to bind to their native ligands (HDL to SR‐B1 and oxLDL to CD36) with high affinity by microscale thermophoresis (MST). However, as SR‐B1 and CD36 are both scavenger receptors, they are able to bind to a variety of lipoprotein ligands and we have demonstrated these interactions with varying binding affinities by MST. Glycosylation of both CD36 and SR‐B1 has been shown to be important for cell surface expression, but the role of glycosylation in ligand binding has remained uncharacterized. We are the first to demonstrate that the glycosylation status of CD36 and SR‐B1 does not alter binding affinities or protein stability. These purified receptors and functional assays provide promise for understanding the ways in which class B scavenger receptors perform their critical functions and lay the groundwork for future structure‐function studies.

PHB2 affects the virulence of Vip3Aa to Sf9 cells through internalization and mitochondrial stability

ABSTRACT The vegetative insecticidal proteins (Vip3A) secreted by some Bacillus thuringiensis (Bt) strains during vegetative growth are regarded as a new generation of insecticidal toxins. Like insecticidal crystal proteins, they are also used in transgenic crops to control pests. However, their insecticidal mechanisms are far less defined than those of insecticidal crystal protein. Prohibitin 2 (PHB2) is a potential Vip3Aa binding receptor identified from the membrane of Sf9 cells in our previous work. In this paper, we demonstrated the interaction between Vip3Aa and PHB2 using pull-down, dot blotting, microscale thermophoresis, and co-immunoprecipitation assays. PHB2 is distributed on the cell membrane and in the cytoplasm, and the co-localization of PHB2 and Vip3Aa was observed in Sf9 cells using a confocal laser scanning microscope. Moreover, PHB2 could interact with scavenger receptor-C via its SPFH (stomatin, prohibitin, flotillin, and HflK/C) domain. Downregulation of phb2 expression reduced the degree of internalization of Vip3Aa, exacerbated Vip3Aa-mediated mitochondrial damage, and increased Vip3Aa toxicity to Sf9 cells. This suggested that PHB2 performs two different functions: Acting as an interacting partner to facilitate the internalization of Vip3Aa into Sf9 cells and maintaining the stability of mitochondria. The latter has a more important influence on the virulence of Vip3Aa.

Synergism of Bacillus thuringiensis Toxin Cry1Ac by a Fragment of Toxin-Binding Polycalin from Plutella xylostella.

The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.

Identification of Spodoptera frugiperda importin alphas that facilitate the nuclear import of Autographa californica multiple nucleopolyhedrovirus DNA polymerase.

Proteins containing nuclear localization signals (NLSs) are actively transported into the nucleus via the classic importin-α/β-mediated pathway, and NLSs are recognized by members of the importin-α family. Most studies of insect importin-αs have focused on Drosophila to date, little is known about the importin-α proteins in Lepidoptera insects. In this study, we identified four putative importin-α homologues, Spodoptera frugiperda importin-α1 (SfIMA1), SfIMA2, SfIMA4 and SfIMA7, from Sf9 cells. Immunofluorescence analysis showed that SfIMA2, SfIMA4 and SfIMA7 localized to the nucleus, while SfIMA1 distributed in cytoplasm. Additionally, SfIMA4 and SfIMA7 were also detected in the nuclear membrane of Sf9 cells. SfIMA1, SfIMA4 and SfIMA7, but not SfIMA2, were found to associate with the C terminus of AcMNPV DNA polymerase (DNApol) that harbours a typical monopartite NLS and a classic bipartite NLS. Further analysis of protein-protein interactions revealed that SfIMA1 specifically recognizes the bipartite NLS, while SfIMA4 and SfIMA7 bind to both monopartite and bipartite NLSs. Together, our results suggested that SfIMA1, SfIMA4 and SfIMA7 play important roles in the nuclear import of AcMNPV DNApol C terminus in Sf9 cells.

Fibroblast Growth Factor Receptor, a Novel Receptor for Vegetative Insecticidal Protein Vip3Aa.

Vegetative insecticidal proteins (Vips), which are secreted by some Bacillus thuringiensis strains during vegetative growth, exhibit high virulence to many pests. Vip3A proteins have been used commercially both in some bio-insecticides and in transgenic crops; however, compared with insecticidal crystal proteins, the mechanism of action of Vip3A is still unclear. In this work, we indicated that the fibroblast growth factor receptor-like protein (Sf-FGFR) from the membrane of Sf9 cells could bind to Vip3Aa. The interaction between Vip3Aa and Sf-FGFR was confirmed by pull-down assays and dot blotting experiment in vitro. The binding affinity between Vip3Aa and extracellular regions of Sf-FGFR (GST-FGFR-N) was determined by microscale thermophoresis assay (MST). Moreover, Vip3Aa-Flag could be co-immunoprecipitated with Sf-FGFR-V5 ex vivo. Furthermore, knockdown of Sf-FGFR gene in Sf9 cells resulted in reducing the mortality of those cells to Vip3Aa. In summary, our data indicated that Sf-FGFR is a novel receptor for Vip3Aa.

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols.

The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transport them was determined using a baculovirus-Sf9 insect cell system. Our results indicate that all thiols tested [GSH, T(SH)2] as well as their oxidized forms GSSG and TS2 (trypanothione disulfide) stimulate LABCG2-ATPase basal activity. We have measured the transport of [3H]-GSH in inside-out Sf9 cell membrane vesicles expressing LABCG2-GFP (green fluorescence protein), finding that LABCG2 was able to mediate a rapid and concentration-dependent uptake of [3H]-GSH in the presence of ATP. Finally, we have analyzed the ability of different thiol species to compete for this uptake, T(SH)2 and TS2 being the best competitors. The IC50 value for [3H]-GSH uptake in the presence of increasing concentrations of T(SH)2 was less than 100 μM, highlighting the affinity of this thiol for LABCG2. These results provide the first direct evidence that LABCG2 is an ABC transporter of reduced and oxidized non-protein thiols in Leishmania, suggesting that this transporter can play a role in the redox metabolism and related processes in this protozoan parasite.

Budded baculovirus particles as a source of membrane proteins for radioligand binding assay: The case of dopamine D1 receptor

IntroductionG-protein-coupled receptors have become very important drug targets and therefore ligand binding assays for these receptors are an essential part of drug discovery. Among a variety of experimental systems, the radioligand binding assay has remained as one of the main methods in this field for decades. Usually cell membranes or tissues are used in these experiments, however in this article we demonstrate that baculoviruses produced in Sf9 cells display recombinant receptors on their surface and therefore can be used in radioligand binding assay. MethodsWe have used baculoviruses with dopamine D1 receptors as a model system to validate the usage of this receptor preparation in radioligand binding experiments. In order to collect membrane receptors and separate free radioligand, we have applied FilterMate Harvester with Filtermat B. ResultsUsing baculoviruses with dopamine D1 receptors as a model system, we have shown that this is a suitable preparation for conventional radioligand binding assay. Here, all the experiments were performed using the dopamine D1-like receptor specific radioligand [3H]SCH23390. There were no significant differences between binding parameters determined in membranes of Sf9 cells and baculovirus particles. Similar pIC50 and Ki values were also determined in competition binding assays with HEK293 cell membranes. DiscussionAll the results obtained with baculovirus preparation were in good agreement with the data obtained in parallel experiments with membrane preparations from Sf9 and HEK293 cells expressing dopamine D1 receptors. Shape uniformity, homogeneous distribution and slow sedimentation of the membranes are some of the advantages of baculovirus preparations, which prove them as promising source of membrane proteins for routine and high throughput analysis.

Nanostructural characterization of Sf9 cells during virus-like particles generation.

Sf9 cells (a clonal isolate of Spodoptera frugiperda Sf21 cells) are commonly used to generate recombinant virus-like particles (VLPs). For VLPs generation, Sf9 cells are infected with recombinant baculoviruses (rBV) expressing desired proteins. During rBV infections, Sf9 cells have changes in cell diameters and surface structures. In this study, for the first time, we investigated nanostructural changes of Sf9 cells using atomic force microscopy (AFM) during VLPs generation containing Toxoplasma gondii rhoptry protein 18 (ROP 18). As results, Sf9 cells were changed to be larger at 2 days after rBV infections. They maintained their sizes and morphologies on day 3 and 4. Based on morphological (perimeter and diameter) and surface roughness (roughness average and root mean square) changes of Sf9 cells observed by AFM, we inferred that these nanostructural changes in Sf9 cell membranes might be due to the production and extrusion of VLPs after rBV infection. Our results suggest that shape and roughness parameters of Sf9 cell morphology and membrane surface by AFM could be very effective for quantitative analysis of VLP production. This study provides important information about structural and mechanochemical properties of Sf9 cells which are closely related with biological function. SCANNING 38:735-742, 2016. © 2016 Wiley Periodicals, Inc.

Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [(35)S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([(35)S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.

Phosphorylation of Synaptic GTPase-activating Protein (synGAP) by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) and Cyclin-dependent Kinase 5 (CDK5) Alters the Ratio of Its GAP Activity toward Ras and Rap GTPases

synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.

(3) H]UR-DE257: development of a tritium-labeled squaramide-type selective histamine H2 receptor antagonist.

A series of new piperidinomethylphenoxypropylamine-type histamine H2 receptor (H2 R) antagonists with different substituted "urea equivalents" was synthesized and characterized in functional in vitro assays. Based on these data as selection criteria, radiosynthesis of N-[6-(3,4-dioxo-2-{3-[3-(piperidin-1-ylmethyl)phenoxy]propylamino}cyclobut-1-enylamino)hexyl]-(2,3-(3) H2 )propionic amide ([(3) H]UR-DE257) was performed. The radioligand (specific activity: 63 Ci mmol(-1) ) had high affinity for human, rat, and guinea pig H2 R (hH2 R, Sf9 cells: Kd , saturation binding: 31 nM, kinetic studies: 20 nM). UR-DE257 revealed high H2 R selectivity on membranes of Sf9 cells, expressing the respective hHx R subtype (Ki values: hH1 R: >10000 nM, hH2 R: 28 nM, hH3 R: 3800 nM, hH4 R: >10000 nM). In spite of insurmountable antagonism, probably due to rebinding of [(3) H]UR-DE257 to the H2 R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H2 R affinities in competition binding assays.

Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs

Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site.

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.

Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: Baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2

The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections.

English

In the present study, one recombinant baculovirus BacSC-GP5, expressing His6-tagged GP5 with the transmembrane domain (TM) and cytoplasmic domain (CTD) derived from baculovirus envelope protein gp64, was constructed and its immunogenicity andprotective efficiency was evaluated in piglets. The results obtained show that, His6-tagged recombinant GP5 was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. Immunogold electron microscopy demonstrated that, the GP5 glycoprotein was displayed successfully on the viral surface.Piglets immunized with BacSC-GP5 induced successfully GP5-specific enzyme-linked immunosorbent assay (ELISA) antibody, neutralizing antibody and lymphocyte proliferation response at 6 weeks after primary immunization. An in vivo challenge result indicated that piglets immunized with BacSC-GP5 did not show any obvious clinical signs and histological changes, and the quantitative real-time polymerase chain reaction (RT-PCR) also indicated that the porcine reproductive and respiratory syndrome virus (PRRSV) viral load from the serum in BacSC-GP5 group was significantly reduced at 14 and 21 days post-challenge compared to that in the negative control group. These results indicate that baculovirus-mediated gene delivery can be utilized as an alternative strategy to develop a new generation of vaccine against PRRSV infection.   Key words: Baculovirus surface display, porcine reproductive and respiratory syndrome virus (PRRSV), GP5 protein, immunogenicity, protective efficacy.

Baculovirus Virions Displaying Infectious Bursal Disease Virus VP2 Protein Protect Chickens Against Infectious Bursal Disease Virus Infection

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.

Water permeability and characterization of aquaporin-11

The water permeability of aquaporin-11 (AQP11), which has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif in the water channel family, is controversial. Our previous study, however, showed that AQP11 is water permeable in proteoliposomes in which AQP11 molecules were reconstituted after purification with Fos-choline 10, which is the most suitable detergent available for stable solubilization of AQP11. In our previous study, we were unable to exclude the effect of the detergent on the water conductance. Therefore, in the present study, we measured the water permeability of AQP11 without detergent using vesicles that directly formed from Sf9 cell membranes expressing AQP11 molecules. The water permeability of AQP11 was 8-fold lower than that of AQP1 and 3-fold higher than that of mock-infected cell membrane, and was reversibly inhibited by mercury ions. Considering the slow but constant water permeable functions of AQP11, we performed homology modeling to search for a common structural feature. When comparing our model with those of other AQP structures, we found that Tyr83 facing the channel pore might be a key amino acid residue that decreases the water permeation of AQP11. Our findings indicate that AQP11 could be involved in slow but constant water movement across the membrane.