[Construction of recombinant baculovirus vectors started by EF1alpha].

OBJECTIVE: To improve the transduction efficiency of baculovirus and exogenous gene expression level, we chose a mammalian cell-specific promoter-human extension factor 1alpha promoter (EF1-alpha), used virus pseudotyped tools--truncated vessicular stomatitis virus protein G (VSV-GED), added woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and adenovirus inverted terminal repeats (ITRs). METHOD: We constructed two improved recombinant baculoviruses transfer vectors named pWK and pWK-ITR with the pFB-VSV-GED-WPRE. The recombinant transfer vectors pWK-eGFP, pWK-ITR-eGFP and pWK (-)-eGFP were constructed by inserting the Enhanced Green Fluorescent Protein (eGFP) reporter gene into the downstream of EF1alpha promoter. Constructed recombinant plasmid transfected Sf9 insect cells, and observed the expression of green fluorescent protein by using the inverted fluorescence microscope. RESULTS: The fluorescence expression rate of BV-WK-eGFP, BV-WK-ITR-eGFP containing WPRE and ITRs was significantly higher than the negative control, ITRs can effectively extend the expression time of eGFP, the expression time of eGFP in BV-WK-eGFP and BV-WK-ITR-eGFP increased 72 hours compared to the negative control BV-WK (-) -eGFP. The transduction time of VSV-GED pseudotyped baculovirus BV-WK-eGFP, BV-WK-ITR-eGFP was obviously shorten in OL cells, and reduced 24 hours compared to the negative control BV-WK (-) -eGFP, transduction efficiency were higher 25.7% and 36.5% than the negative control BV-WK (-) -eGFP, respectively. CONCLUSION: The experiments proved that the VSV-GED could effectively improve the transduction efficiency of baculovirus, WPRE could enhance the expression efficiency of the exogenous gene significantly, and ITRs extend the expression time. The research will lay a foundation to explore improved recombinant baculovirus express exogenous genes in vertebrate cells and research the new recombinant live vector vaccine.

[Construction of baculovirus vector with Cytomegaloviruse promoter to express eGFP in primary chicken embryo cells].

OBJECTIVE: Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells. The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter, VSV-GED and different functional regulatory elements. The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells, which provides us a new approach to develop engineered poultry vaccines. METHOD: The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor, VSV-GED, woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs). The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells, and the eGFP protein expression levels mediated by different baculoviruses were analyzed. RESULTS: The expression of eGFP was detected at 12 hours after infection. The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED. The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10 mmol/L butyrate. However, the WPRE elements are nontoxic to cells. Within 72 hours, the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually. CONCLUSION: The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells. The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.

[Construction of BV-T7 hybrid expression system based on baculovirus to express target gene eGFP in mammalian and chicken cells].

OBJECTIVE: To investigate whether the recombinant baculovirus BV-T7 hybrid expression system can be effectively transduced into chicken cells in vitro, and whether it can express the foreign genes (eGFP). METHOD: We established a hybrid baculovirus-T7 RNA polymerase system for transient expression in mammalian cells and chicken cells. Two recombinant baculoviruses were constructed, one carrying cDNA of bacteriophage T7 RNA polymerase, with a nuclear localization signal, under the control of a mammalian promoter and the other expressing eGFP gene under the control of T7 promoter. The constructed recombinant baculoviruses co-infected mammalian oligodendrocyte cells, as well as chicken embryo fibroblasts cells and chicken embryo skeletal muscle cells. RESULTS: The eGFP activity was detected in mammalian cell lines and embryo fibroblasts cells and chicken embryo skeletal muscle cells. The recombinant baculovirus transduction efficiency of oligodendrocyte cells was 59.5%, and in CEF cells and myoblast cells the transduction efficiencies were 23.2% and 33.1%. CONCLUSION: BV-T7 hybrid expression could be expressed T7 RNAP in mammalian cells and avian cells.

[The expression of IBDV VP2 in chicken primary myoblast cells using the baculovirus vector].

OBJECTIVE: To construct the recombinant baculovirus expressing Infectious bursal disease (IBDV) VP2 gene in the chicken primary myoblast cells. METHODS: A proteinase K digestion and phenol-chloroform extraction method was used to extract dsRNA genome from IBDV. VP2 gene was amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR) with the genome RNA as template. The pFastBac-pCMV-VP2 baculovirus transfer vector was constructed by inserting VP2 gene under the immediate-early promoter of cytomegalovirus. The VP2 recombinant bacmid was obtained by Bac-to-Bac system and transfected sf9 insect cell to acquire VP2 recombinant baculovirus. After amplification of recombinant baculovirus on cell passages, the recombinant virus was seeded on chicken primary myoblast cells with 50 multiplicity of infection (MOI), and the cells were harvested at 72 hours after infection. RESULTS: Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) and Western blot results showed that the VP2 gene was successfully expressed in chicken primary myoblast cells. The product was a 48kDa protein and could be recognized by anti-IBDV serum. CONCLUSION: The recombinant baculovirus could efficiently delivery IBDV VP2 gene into chicken primary cells and that CMV, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct the expression of VP2 antigen protein. The research can be a potential basis for the development of baculovirus vector vaccines for IBDV and other avian infectious disease.

Baculovirus-mediated gene expression in chicken primary cells.

A recombinant baculovirus was constructed containing an expression cassette with a reporter gene, green fluorescent protein, directed by a constitutive mammalian promoter: a human cytomegalovirus immediate early promoter/enhancer (CMV-IE). High titer virus was prepared with ultracentrifugation. Efficient gene delivery and expression were observed in the virus-treated chicken primary culture, myoblast cells, and whole embryonic fibroblast cells. It was noticed that an addition of sodium butyrate (a selective histone deacetylase inhibitor) to viral transduction medium extremely enhanced the reporter-gene expression. However, there is no effect of presence of trichostatin A observed. To maximize the reporter-gene expression, the baculoviral infection condition was optimized with both cell types. Our approaches demonstrated that recombinant baculovirus could efficiently deliver its genome DNA into chicken primary cells and that CMV-IE, a mammalian-cell-active promoter, was functional in chicken primary cells and could direct a high level of gene expression. Clearly, the recombinant baculovirus provides an alternative means for foreign gene delivery into avian cells.

Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT2C receptor isoforms.

In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine-serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT2) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT2 receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT2C receptor (VNI isoform) mediated calcium signaling (EC50 1070 nM, IC50 281 nM). It exhibited no appreciable 5-HT2A or 5-HT2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT2A or 5-HT2B receptor expressed in CHO cells (IC50s of 369 and 0.46 nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT2A (IC50, 2.5 nM)>5-HT2B (47 nM)>5-HT2C (69 nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC50s of 571, 1086 and 2099 nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC50s of 1138 and 1000 nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC50 594 nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC50, 79 nM)>VSV (101 nM)>INI (3856 nM). The modest 5-HT2A antagonism and 5-HT2C partial agonism, along with reported D2 and 5-HT1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine-serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT2C agonism may also underlie the minimal weight gain seen with aripiprazole.

Ligand dependency of 5-hydroxytryptamine 2C receptor internalization.

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT)2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT2C receptor agonists serotonin, Ro 60-0175 [(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine], and WAY-161503 [(4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one]; partial agonists mCPP [1-(m-chlorophenyl)piperazine] and DOI [(+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]; inverse agonists SB-206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide] and mianserin; and neutral antagonists SB-242084 [6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline] and 5-methoxygramine on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca2+ measurements (flourometric imaging plate reader). The 5-HT2C-GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in approximately 50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.