ABSTRACT
OBJECTIVE@#To investigate the anti-tumor effects and the mechanism of the recombinant fowlpox virus expressing Apoptin gene on human laryngeal carcinoma Hep-2.@*METHOD@#Hep-2 cells cultured in vitro were infected with vFVApoptin. The anti-tumor effects on Hep-2 cells were measured through MTT staining and, the mitochondrial trans-membrane potential (delta psi m) and reactive oxygen species (ROS) were analyzed by flow cytometry. Western blot was used to detect the release of cytochrome c (Cyto c). Caspase-3/9 activities were measured by colorimetric assay.@*RESULT@#vFVApoptin could restrain Hep-2 cells significantly and, had the function of down-regulating delta psi m, up-regulating ROS, promoting Cyto c release and activating Caspase-3/9.@*CONCLUSION@#Cyto c were released from mitochondria by the function of up-regulating ROS of vFVApoptin. Cyto c triggered Caspase-9 and, after the activation of Caspase-9, downstream apoptotic factors, such as caspase-3, were activated. Eventually, Hep-2 cells were suppressed by mitochondrial pathway apoptosis induced by vFVApoptin.
Subject(s)
Animals , Humans , Apoptosis , Capsid Proteins , Genetics , Pharmacology , Chicken anemia virus , Genetics , Fowlpox virus , Genetics , Tumor Cells, CulturedABSTRACT
The hemagglutinin (HA) gene from H5N1 avian influenza virus and the chicken interleukin 2 (chiIL-2) gene were inserted into a expressing vector p12LS to construct a recombinant transferring vector p12LSH5AIL2, in which HA gene under the control of the promoter Ps was in inverse tandem connection with the chiIL-2 gene under the control of the promoter PE/L. The p12LSH5AIL2 was then used to transfect the chicken embryo fibroblasts (CEF) pre-infected with a wild-type fowlpox virus 282E4 strain, to generate a recombinant fowlpox virus coexpressing the inserted HA and chiIL2 genes (rFPV-H5AIL2). The rFPV-H5AIL2 was obtained and purified by blue plaque screening on the CEF. The in vitro expression of HA gene by rFPV-H5AIL2 was detected in the recombinant fowlpox virus-infected CEFs with an indirect immunofluorescence assay, and the expression of the chiIL2 gene by rFPV-H5AIL2 was confirmed by detection of the chiIL2 mRNA by RT-PCR and by detection of chiIL2 by the indirect immunofluorescence assay. Experiments on SPF and commercial chickens demonstrated that the titer for HI antibodies induced by the rFPV-H5AIL2 was significantly higher than that by the rFPV-HA. The group immunized with the rFPV-H5AIL2 exhibited the similar ratios of protective efficacy and virus shedding as the group immunized with the rFPV-HA in SPF chicken. However, in commercial chicken, the group immunized with the rFPV-H5AIL2 generated significantly higher protection against H5N1 avian influenza virus challenge and lower virus shedding than the group immunized with the rFPV-HA. This study paved the way for further development of a new AIV recombinant vaccine.
Subject(s)
Animals , Chick Embryo , Cells, Cultured , Chickens , Fowlpox virus , Genetics , Metabolism , Gene Expression , Genetic Engineering , Genetic Vectors , Genetics , Metabolism , Hemagglutinins , Genetics , Allergy and Immunology , Influenza A Virus, H5N1 Subtype , Genetics , Allergy and Immunology , Influenza in Birds , Allergy and Immunology , Virology , Interleukin-2 , Genetics , Allergy and Immunology , Random AllocationABSTRACT
The CSFV E0 gene was amplified from the plasmid pMD18-T-E0 by PCR and cloned into the FPV-P11 and FPV-pSY. The identified recombinant DNA was transfected into chicken embryo fibroblasts (CEF) to package Fowlpox virus. E0 gene was confirmed to be integrated into the genome of recombinant Fowlpox virus by PCR, and Western blot was employed for detection of E0 expression in the chicken embryo fibroblasts infected with recombinant Fowlpox virus . The results of ELISA showed that systemic immune response to CSFV could be induced effectively after the mice were immunized three times with recombinant Fowlpox virus through celiac route, the titer of antibody was 1 : 4096. The protection experiment showed that 75% of piglets immunized three times with recombinant Fowlpox virus were survived, indicating that the recombinant Fowlpox virus was effective. This paper lays foundation for the study of CSFV live vector vaccine.
Subject(s)
Animals , Chick Embryo , Female , Mice , Blotting, Western , Classical Swine Fever Virus , Genetics , Allergy and Immunology , Enzyme-Linked Immunosorbent Assay , Fowlpox virus , Genetics , Mice, Inbred BALB C , Polymerase Chain Reaction , Swine , Vaccines, Synthetic , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and Immunology , Viral Vaccines , Allergy and ImmunologyABSTRACT
The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2) while the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ were characterized by Western blot (F and gB proteins) and indirect immunofluorescence test (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10 kb gene fragment, could be expressed authentically and efficiently. Compared to the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken embryo fibroblasts (CEF) cell culture. Overall, our work suggests that FPV can be a useful live virus vector for the expression of multi- foreign genes against multiple avian pathogens.
Subject(s)
Animals , Cloning, Molecular , Fibroblasts , Virology , Fowlpox virus , Genetics , Gene Expression , Genetic Engineering , Methods , HN Protein , Genetics , Herpesvirus 1, Gallid , Genetics , Physiology , Newcastle disease virus , Genetics , Physiology , Plasmids , Genetics , Transfection , Viral Envelope Proteins , Genetics , Viral Fusion Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.</p><p><b>METHODS</b>The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.</p><p><b>RESULTS</b>There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.</p><p><b>CONCLUSION</b>The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.</p>
Subject(s)
Animals , Chick Embryo , Mice , Antibodies, Viral , Blood , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fibroblasts , Cell Biology , Metabolism , Fowlpox , Blood , Allergy and Immunology , Virology , Fowlpox virus , Genetics , Allergy and Immunology , Gene Products, gag , Genetics , Metabolism , Genetic Vectors , Genetics , HIV Envelope Protein gp120 , Genetics , Metabolism , HIV-1 , Genetics , Metabolism , Immunization , Methods , Interleukin-6 , Genetics , Metabolism , Mice, Inbred BALB C , Microscopy, Electron , Plasmids , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , Transfection , Viral Vaccines , Genetics , Allergy and Immunology , MetabolismABSTRACT
In order to determine the adjuvant effects of the chicken IL-2 (ChIL-2) on new generation vaccines, ChIL-2 gene was amplified from ConA-stimulated chicken spleen cells by RT-PCR and was directionally inserted into fowlpox virus (FPV) transferring vector p1175 under the control of FPV early/late promoter (PE/L), resulting in recombinant transferring vector p1175IL2. Then the p1175IL2 plasmid was transfected into chicken embryo fibroblasts (CEF) pre-infected with wild type FPV to generate recombinant fowlpox virus expressing ChIL-2 (rFPV-IL2). By selection of blue plaques on the CEF, overlaid with agar containing X-gal, rFPV-IL2 was obtained and purified. The supernatant from CEF monolayer infected with rFPV-IL2 (M.O.I2.0) after 72 hours was detected for the production of ChIL-2 by XTT/PMS colorimetric assay. About 3.6 x 10(5) u/mL of specific ChIL-2 activity was determined. The results show that rFPV-IL2 can express ChIL-2 effectively. rFPV-IL2 provides us with an effective tool for studying avian immunology as well as a potential vaccine-enhancing agent.
Subject(s)
Animals , Chick Embryo , Chickens , Fowlpox virus , Genetics , Interleukin-2 , Genetics , Pharmacology , Recombinant Proteins , PharmacologyABSTRACT
To screening out Chinese vaccine candidate against HIV-1, Chinese vaccine strain 282E4 of fowlpox virus was used as the vector to construct the recombinant fowlpox virus (rFPV) coexpressing gp120 of Chinese HIV-1 strain and IL-18, and the recombinant virus was indentified by PCR and Western blot. The specific DNA fragment could be amplified by PCR from the genome of rFPV. Western blot analysis showed that gp120 and IL-18 could be expressed not only in chicken embryo fibroblast (CEF) cells infected by rFPV, but also in mammalian cells infected by rFPV. After the recombinant fowlpox virus was inoculated into BALB/c mice, the spleen specific CTL activities and serum antibodies in the immunized mice were detected, which demonstrated that the rFPV had good immunogenicity and could induce BALB/c mice to produce specific humoral and cellular immunity. IL-18 palyed the role of immunoadjuvant. The study lays the basis on the preparation of genetic engineering live vector vaccine against HIV-1.
Subject(s)
Animals , Humans , Mice , AIDS Vaccines , Allergy and Immunology , Adjuvants, Immunologic , Antibodies, Viral , Allergy and Immunology , Fowlpox virus , Genetics , Allergy and Immunology , Metabolism , Gene Transfer Techniques , Genetic Vectors , HIV Envelope Protein gp120 , Genetics , Allergy and Immunology , HIV-1 , Genetics , Allergy and Immunology , Immunization , Interleukin-18 , Genetics , Allergy and Immunology , Mice, Inbred BALB C , Vaccines, DNA , Allergy and ImmunologyABSTRACT
Fowlpox virus vaccine strain and two field isolates collected from out breaks of disease were purified from cell culture using sucrose gradient ultracentrifugation. The viral DNAs were digested with Bgl I, Bam HI, Hha I and Sma I restriction endonucleases and the fragment pattern was analysed on 0.7% agarose gel. Bgl I digestion produced 54 fragments of size ranging from 31.50 to 0.60 kb, having similar electrophoretic mobilities in both the vaccine strain and two field isolates. Only 9 well resolved and one unresolved or partially digested fragment were obtained after Bam HI digestion. A total number of 29 and 41 fragments were obtained with Hha I and Sma I respectively. Almost similar restriction fragment pattern was observed in vaccine strain and the field isolates. The total genomic size was calculated to be between 265.00 and 302.91 kb. The three viruses were found to be genetically similar.
Subject(s)
Animals , Chick Embryo , DNA Restriction Enzymes , DNA, Viral/genetics , Fowlpox virus/genetics , Polymorphism, Restriction Fragment Length , PoultryABSTRACT
A fowlpox virus isolate obtained from an outbreak of disease in a vaccinated poultry flock was propagated in chicken embryo fibroblast cell culture. Analysis of purified virus polypeptide on 7.5-15% gradient polyacrylamide gel revealed 45 structural polypeptides after Coomassie blue staining. The mol.wt. of polypeptides ranged between 225.53 and 10.50 kDa with total mol.wt. of 2650 kDa. Variable numbers of immunogenic virion polypeptides were detected in immunoblot with fowlpox virus infected chicken sera collected at different time intervals. A total of 29 polypeptides reacted with sera collected at 1st week post-infection and the number gradually declined to 27, 26, 20, 17 and 15 when reacted with 2nd, 3rd, 4th, 5th and 6th week post-infection sera, respectively. Reaction with fowlpox virus hyperimmune sera revealed 35 immunogenic polypeptides. A number of major and minor immunogens were detected. Antisera against seven major single band polypeptides including one double band polypeptide showed very low reactivity both in ELISA and serum neutralization test. Involvement of multigenic components in virus neutralization is indicated.
Subject(s)
Animals , Cells, Cultured , Chick Embryo , Fowlpox virus/immunology , Peptides/analysis , Vaccines, Synthetic/immunology , Viral Proteins/immunologyABSTRACT
Chickens infected with fowlpox virus (FPV) IVRI vaccine strain and two field isolates collected from clinical cases of disease (Bareilly isolate and Panchmahal isolate) produced humoral antibody response after 2nd week post-infection, with a noticeable variation in degree of immune response. Serum antibody titre peaked at 4th week post-infection with a titre of 25,600, 25,600 and 51,200 being detected in ELISA and neutralization index of 2.75, 2.43 and 3.12 in serum neutralization test (SNT) with IVRI vaccine strain, Panchmahal isolate and Bareilly isolate, respectively. Cellular immune response was detected as early as 1st week post-infection by leukocyte migration inhibition test (LMIT). Per cent migration inhibition too peaked at 4th week with a value of 40.30 +/- 3.45, 36.93 +/- 4.11 and 45.45 +/- 3.66 being detected with the three viruses respectively. The Hind III and Hae III restriction fragment profile of viral DNA showed almost similar pattern both in vaccine strain and two field isolates. Hind III digestion produced 47 well resolved fragments of sizes between 24.30 and 1.20 kb and the total genomic size was estimated to be between 305.81 and 306.06 kb. Hae III digestion revealed 34 well resolved fragments of sizes between 27.55 and 1.32 kb. The three viruses could not be differentiated on the basis of their genomic restriction pattern. However immunogenic and antigenic differences were noticed by ELISA and SNT tests.
Subject(s)
Animals , Cells, Cultured , Chick Embryo , Chickens , Fowlpox virus/genetics , Genome, Viral , Immunization , Viral Vaccines/immunologyABSTRACT
Structural polypeptides of IVRI vaccine strain and two field isoaltes of Fowlpox virus (Bareilly isolate and Panchmahal isolate) were analysed on SDS-PAGE and by immunoblotting technique. In 5%-20% gradient acrylamide gel 31, 29 and 31 polypeptide bands and in 7.5%-15% gradient gel 45, 37 and 39 polypeptide bands were detected after Coomassie blue staining respectively for Bareilly isolate, Panchmahal isolate and IVRI vaccine strain. The molecular weight (MW) of the polypeptides ranged from 226.10 to 10.30 kDa with total MW of 2650.12, 2259.50, and 2378.68 kDa respectively for the three viruses. The immunoblot revealed 35, 29 and 30 immunogenic polypeptides indicating most virion polypeptides to be immunogenic in nature. Although most polypeptides had similar electrophoretic pattern both in SDS-PAGE and immunoblotting, still the three viruses could be differentiated. The viruses were found to be antigenically different with Panchmahal isolate lacking the polypeptides 81.15, 76.33, 39.30, 37.50 and 29.35 kDa and the vaccine strain lacking 76.33, 37.50 and 29.35 kDa polypeptides as were present in Bareilly isolate.