Anti-tumor effect of Sendai virus Tianjin strain defective interfering particles on tumor-bearing mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the anti-tumor effect and its mechanism of Sendai virus Tianjin strain defective interfering particles (DIP) on mouse models of colon carcinoma.</p><p><b>METHODS</b>CT26 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.</p><p><b>RESULTS</b>On 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.</p><p><b>CONCLUSIONS</b>Tianjin 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.</p>

Detection of replication-defective hepatitis A virus based on the correlation between real-time polymerase chain reaction and ELISA in situ results

ELISA in situ can be used to titrate hepatitis A virus (HAV) particles and real-time polymerase chain reaction (RT-PCR) has been shown to be a fast method to quantify the HAV genome. Precise quantification of viral concentration is necessary to distinguish between infectious and non-infectious particles. The purpose of this study was to compare cell culture and RT-PCR quantification results and determine whether HAV genome quantification can be correlated with infectivity. For this purpose, three stocks of undiluted, five-fold diluted and 10-fold diluted HAV were prepared to inoculate cells in a 96-well plate. Monolayers were then incubated for seven, 10 and 14 days and the correlation between the ELISA in situ and RT-PCR results was evaluated. At 10 days post-incubation, the highest viral load was observed in all stocks of HAV via RT-PCR (10(5) copies/mL) (p = 0.0002), while ELISA revealed the highest quantity of particles after 14 days (optical density = 0.24, p < 0.001). At seven days post-infection, there was a significant statistical correlation between the results of the two methods, indicating equivalents titres of particles and HAV genome during this period of infection. The results reported here indicate that the duration of growth of HAV in cell culture must be taken into account to correlate genome quantification with infectivity.

Construction and immunogenicity analysis of recombinant replication-defective human adenovirus type 5 bearing the porcine circovirus type 2 Cap protein gene / 病毒学报

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.

Hepatitis B virus core antigen as a carrier for virus-like partical vaccine: a review / 病毒学报

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.

Construction of replication-deficient recombinant adenovirus vector with hTFPI-2 gene by AdMax system and expression in U937 monocytes in vitro / 生物医学工程学杂志

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.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric â-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Construction of replication-deficient recombinant adenoviral vector carrying HBsAg and HSP70 chimeric gene and its expression in vitro / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro.</p><p><b>METHODS</b>Heat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro.</p><p><b>RESULTS</b>The adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 x 10(12) pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection.</p><p><b>CONCLUSION</b>The recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.</p>

Construction and characterization of hSef recombinant adenoviral vectors / 生物工程学报

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.

High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Suicide 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.</p><p><b>METHODS</b>pAdTrack-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.</p><p><b>RESULTS</b>Comet-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.</p><p><b>CONCLUSION</b>The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.</p>

Adenovirus-mediated double suicide gene therapy for experimental bladder carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSIONS</b>Adenovirus 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.</p>

Construction of a recombinant adenovirus vector for interferon-gamma-inducible protein 10 and the adenovirus preparation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector for expressing interferon-gamma-inducible protein 10 (IP-10) by homogenous bacterial recombination.</p><p><b>METHODS</b>IP-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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>A 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.</p>

Characterization and E protein expression of mutant strains during persistent infection of KN73 cells with Japanese encephalitis virus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the character of mutants originating from Japanese encephalitis viruses and the relationship between the characterization of mutant strains and E protein expression.</p><p><b>METHODS</b>Persistent 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSIONS</b>The 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.</p>

Electron microscopic study of phages and aberrant structures produced by induction of prophage kappa in Vibrio cholerae el tor cells.

Prophage kappa in V. cholerae el tor strain SLH22(J) could be induced spontaneously or by treatment with nitrofurantoin, though the efficiency of induction was very low (not more than 0.8%). V. cholerae el tor cells were found to release many different aberrant structures of the temperate phage, kappa. These aberrant structures were characterized by density gradient centrifugation and electron microscopy.