Hepatitis B virus core antigen (HBcAg) is a major viral nucleocapsid protein of HBV. It is a 21-22kD protein consisting of 183-185 amino acids. Because of its easy purification, strong immunogenicity, high expression level, and self-assembles into the virus-like particles (VLP), HBcAg could be an efficient and safe VLP carrier for developing vaccines for various pathogens. Up to now, HBcAg VLP carrier has been an important system to develop novel vaccines and many antigen epitope genes from viruses, bacteria and parasites were expressed successfully using the system.
Animals ; Defective Viruses ; genetics ; immunology ; Gene Expression ; Hepatitis B Core Antigens ; genetics ; immunology ; Humans ; Vaccines ; genetics ; immunology

We tried to construct and identify the recombinant replication-deficient adenovirus vector coding for human tissue factor pathway inhibitor 2 (hTFPI-2) gene by AdMax system in HEK293 cells. Firstly, we obtained hTFPI-2 gene from the recombinant plasmid pIRES2-EGFP-TFPI-2 by PCR using primers with restriction endonuclease site of EcoRI or SacI. After digesting the hTFPI-2 gene and plasmid PDC316-IRES-EGFP shuttle vector, we ligated them with T4 ligase and formed the recombinant shuttle vector PDC316-IRES-EGFP-hTFPI-2. It was confirmed that the ligation product was inserted the gene of hTFPI-2 correctly by sequencing. Then we took cotransfection of HEK293 cells with the recombinant shuttle vector and genomic plasmid pBHGloxdeltaE1,3Cre by liposome lipofectamine2000, and finished the package of recombinant adenovirus Ad-hTFPI-2. The results of the PCR test and restriction endonuclease digestion confirmed the successful construction of the recombinants Ad-hTFPI-2. Furthermore, we measured the titre of Ad-hTFPI-2 with the aid of green fluorescence protein expression after multiplication and purification. The titre was 0.931 x 10(12) pfu/ml. Finally, we infected U937 monocytes by purified Ad-hTFPI-2, and determined the infection efficiency and the TFPI-2's level and activity. The efficiency of Ad-hTFPI-2 infection in U937 cells was 89.33%. After infected by Ad-hTFPI-2, the TFPI-2's level in supernatant increased about 7 fold. Also the TFPI-2 in supernatant had activities of inhibiting trypsin and plasmin. The recombinant adenovirus with the hTFPI-2 gene was constructed successfully. It will be helpful for the further investigation of its potentiality to be applied in antiatherosclerosis.
Adenoviridae ; genetics ; metabolism ; Defective Viruses ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Monocytes ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; U937 Cells

To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up to 10(8.5) TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 10(7) TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.
Adenoviruses, Human ; genetics ; Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Circovirus ; immunology ; Defective Viruses ; genetics ; HEK293 Cells ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; Virus Replication

BACKGROUNDSuicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene (tk)-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses.

METHODSpAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease PacI and polymerase chain reaction (PCR), plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with PacI and sequently transfected into human embryo kidney 293 cells (HEK293) using Lipofectamine 2000.

RESULTSComet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture. After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk.

CONCLUSIONThe replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Adenoviridae ; genetics ; Bacteria ; genetics ; Defective Viruses ; genetics ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; Recombination, Genetic ; Thymidine Kinase ; genetics ; Virus Replication

OBJECTIVETo study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.

METHODSPersistent infection was established with standard strains of Japanese encephalitis viruse, known as parental viruse, in a human hepatoma cell line, KN73. Cells were subcultured weekly using trypsinization techniques. Cell-associated viruses of persistently infected cells were collected by a freeze and thaw method. Virus titers were examined by plaque method using baby hamster kidney (BHK) cells. Indirect immunofluorescence assays were used to examine E and NS3 protein antigens. Western blot analysis was used to test expression of E and NS3 proteins.

RESULTSIn the early phase (24 - 36 h) post-infection, virus titer in culture fluid from KN73 cells infected with parental viruses were 10(6) PFU/ml. They were 10(3 - 4) PFU/ml in the late phase (3 years) post-infection. The titer of cell-associated viruse was 10(2 - 3) PFU/ml. A virus super-infection assay found that virus titers in culture fluid from persistently infected KN73 cells acutely super- infected with parental viruses were much lower than that of culture fluids in acutely infected normal KN73 at the same phase. Indirect immunoflurescence assay revealed that the quantity of viral antigens in persistently infected KN73 cells was lower than that in acutely infected KN73 cells with parental viruses. Western blot analyses indicated that the molecular weights of E and NS3 proteins were 53 kD and 73 kD, respectively. Expression of NS3 protein in persistently infected KN73 cells was stable but expression of E protein was markedly suppressed.

CONCLUSIONSThe virulence and reproduction of viruses obtained from persistently infected KN73 cells, which have some features of DI viruses and were involved in persistent infection, was lower than that of parental viruses. These mutants may have be related to the decrease in E protein expression.

Carcinoma, Hepatocellular ; virology ; Defective Viruses ; physiology ; Encephalitis Virus, Japanese ; chemistry ; genetics ; physiology ; Humans ; Membrane Glycoproteins ; analysis ; Mutation ; RNA Helicases ; Serine Endopeptidases ; Tumor Cells, Cultured ; Viral Envelope Proteins ; analysis ; Viral Nonstructural Proteins ; analysis

OBJECTIVETo construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination.

METHODSIP-10 gene was cloned into the shuttle plasmid pAdTrack-CMV that contained the coding sequence of enhanced green fluorescent protein (EGFP). The shuttle plasmid was then transformed into E. coli BJ5183 with pAdEasy-1 vector by chemical transformation. The recombinant adenovirus vector pAd/IP-10 was identified by enzyme digestion with Pac I and the linearized plasmid was transfected into HEK293 cells.

RESULTSThe positive clones were identified with enzyme digestion and polymerase chain reaction (PCR) and were further verified by DNA sequencing. The recombinant adenovirus of high titration was obtained after transfection and packaging in HEK293 cells.

CONCLUSIONA recombinant adenovirus vector for expression of IP-10 has been constructed successfully and high-titer active adenovirus is obtained for functional study of IP-10 protein.

Adenoviridae ; genetics ; Cell Line ; Chemokine CXCL10 ; genetics ; metabolism ; Cloning, Molecular ; Defective Viruses ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Virus Cultivation ; methods

Defective Viruses ; metabolism ; Epstein-Barr Virus Infections ; diagnosis ; prevention & control ; therapy ; Herpesvirus 4, Human ; isolation & purification ; Histiocytosis, Non-Langerhans-Cell ; diagnosis ; Humans ; Infectious Mononucleosis ; diagnosis ; Leukoplakia, Hairy ; diagnosis ; Lymphoproliferative Disorders ; diagnosis ; Vaccination ; Virus Latency

Sef (similar expression to fgf genes) was identified as a feedback antagonist of FGF signaling in zerbrafish, mouse and human. To construct recombinant adenoviral vectors expressing hSef-L and hSef-S, the coding sequences of the two isoforms were amplified and ligated into pAdTrack-CMV, forming shuttle vectors pAdTrack-CMV/hSef-L-Myc and pAdTrack-CMV/hSef-S-Myc. After sequence confirmation, these two shuttle vector plasmids were linearized by Pme I and then co-transformed respectively with the adenoviral genome vector pAdEasy-1 into E. coli BJ5183. The successful recombinants were selected by Kanamycin and confirmed by Pac I digestion. The recombinant vectors Ad-hSef-L-Myc and Ad-hSef-S-Myc were finally digested with Pac I and transfected into HEK293 cells to pack into viral particles. The virus were amplified in 293 cells and used to infect MEF cells. Western blotting analysis was used to demonstrate the expression of hSef-L-Myc and hSef-S-Myc proteins. The inhibitory effects of the adenovirus mediated Sef expression on FGF signaling was further evaluated by Elk luciferase reporter assay. Our results indicated the constructed virus could produce effectively the proteins and then inhibit FGF signaling in MEF cells.
Adenoviridae ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Defective Viruses ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Protein Isoforms ; biosynthesis ; genetics ; Receptors, Interleukin ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; Virus Cultivation ; methods

OBJECTIVETo explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles (DIP) on mouse models of colon carcinoma.

METHODSCT26 cells (5×10(6)/0.1 ml) were subcutaneously injected into the back of Bal B/c mice to establish murine colon carcinoma model. After the tumors reached 5 mm in diameter, the mice were randomly divided into Tianjin strain DIP group and saline control group. The former was intratumorally injected with Tianjin strain DIP (0.1 ml) once a day on day 4, 7, 10 and 13 after CT26 cell inoculation. The latter was intratumorally injected with the same volume of saline. Tumor volume and survival rate of the mice were calculated to confirm the anti-tumor effect of DIP. Flow cytometry and ELISA were used to examine the maturation and release of cytokines IL-6, IFN-α and TNF-α from murine myeloid dendritic cells (DCs) induced by Tianjin strain DIP. Moreover, real-time RT-PCR and immunohistochemistry were performed to identify whether the Tianjin strain DIP could induce infiltration of CD11c(+) DCs, CD4(+) and CD8(+) T cells in the tumors.

RESULTSOn day 22 after CT26 cell inoculation, the average tumor volume of the Tianjin strain DIP group was (33.2 ± 2.0) mm(3), significantly smaller than that of the control group [(2 376.0 ± 130.8)mm(3), P < 0.01]. On day 50 after CT26 cell inoculation, the survival rate of mice was 90.0% in the Tianjin strain DIP group, much higher than that of the control group (30.0%, P < 0.01). Flow cytometry analysis showed that the expression of markers of DCs maturation, including CD40, CD80 and CD86, was dose-dependently increased by DIP or intact virus. No statistically significant difference was found betweent the DIP and intact virus groups. ELISA results showed that DIP could stimulate the secretion of IL-6, IFN-α and TNF-α from mouse DCs. The secretion of all of the cytokines was dose-dependently increased by DIP or intact virus. Real-time RT-PCR revealed that the expression of CD4, CD8 and CD11c mRNAs was increased in tumors treated with DIP compared with that of the saline group at all time points. Moreover, the expression level of all of them remained maximal at 120 h after the last treatment. Immunohistochemical staining revealed that the ratios of CD4(+), CD8(+) T cells or CD11c(+) DCs to total cells were (21.60 ± 1.49)%, (22.12 ± 2.84)% and (23.05 ± 2.91)%, respectively, in the DIP-treated tumors. In the tumors treated by saline, the ratios were (2.62 ± 0.60)%, (4.05 ± 0.12)% and (3.10 ± 0.09)%, respectively. The difference between experimental group and control group had statistical significance.

CONCLUSIONSTianjin strain DIP may exert anti-tumor effect on tumor-bearing mice. The mechanism is related with the antitumor immunity induced by DCs and T cells.

Animals ; Cell Line, Tumor ; Colonic Neoplasms ; metabolism ; pathology ; Cytokines ; metabolism ; Defective Viruses ; immunology ; Dendritic Cells ; metabolism ; Female ; Interferon-alpha ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Random Allocation ; Sendai virus ; immunology ; T-Lymphocytes ; metabolism ; Tumor Burden ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo evaluate the feasibility and efficacy of cytocine deaminase-thymidine kinase (CD-TK) fusion double suicide gene therapy using adenovirus mediated CD-TK gene and green fluorescent rotein (GFP) gene combined with ganciclovir(GCV) or 5-flourocytosine(5-FC) in a murine subcutaneous bladder carcinoma model.

METHODSA replication defective adenovirus vector containing CD-TK gene was used. Subcutaneous tumors were established in syngenic C57BL/6 female mice with 1 x 10(6) Mb49 cells. Intratumoral injection of AdCD-TK (1.58 x 10(8) PFU, qd x days) in combination with GCV (40 mg.kg(-1).d(-1), ip, qd x 10 days) or 5-FC (400 mg.kg(-1).d(-1), ip, qd x 10 days) was administered in vivo for the determination of treatment efficacy in separate controlled experiments.

RESULTSIn vivo experiments demonstrated that the mean volume of tumor in the group of AdCD-TK/GCV(326.58+/-109.56 mm(3)), AdCD-TK/5-FC (235.33+/-62.94 mm(3)) and AdCD-TK/(GCV+5-FC) (23.58+/-6.78 mm(3)) was reduced significantly compared with that of control group (993.51+/-158.32 mm(3)) (P=0.00), the mean volume of tumor in the group of AdCD-TK/(GCV+5-FC) was significantly less than that in the group of AdCD-TK/GCV or AdCD-TK/5-FC (P=0.04). Tumor necrosis was revealed by histomorphology compared with control animals.

CONCLUSIONSAdenovirus mediated CD-TK double suicide gene combining with GCV or 5-FC could provide an effective therapy in an experimental murine bladder carcinoma by significantly inhibiting tumor growth. The treatment efficacy of AdCD-TK combining GCV and 5-FC was superior to that of AdCD-TK combining GCV or AdCD-TK combining 5-FC.

Adenoviridae ; genetics ; Animals ; Cell Line ; Cell Line, Tumor ; Cytosine Deaminase ; genetics ; metabolism ; Defective Viruses ; genetics ; Female ; Flucytosine ; pharmacology ; therapeutic use ; Ganciclovir ; pharmacology ; therapeutic use ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Thymidine Kinase ; genetics ; metabolism ; Treatment Outcome ; Urinary Bladder Neoplasms ; pathology ; therapy