Construction and identification of helper-dependent adenoviral vector encoding hemagglutinin protein of swin-origin influenza virus A H1N1 / 生物医学工程学杂志

Abstract: In order to investigate immune protection against swine-origin influenza virus (S-OIV) A H1N1, the helper-dependent adenovirus vector (HDAd) system was exploited to construct recombinant HDAd encoding hemagglutinin (HA). The HA gene was synthesized and cloned to the HDAd backbone. Then, the HDAd/HA DNA molecules were transfected into 293Cre4 cells with calcium phosphate. The cells were infected by helper virus 16 hours after the transfection. The 293Cre4 cells were coinfected with HDAd/HA and the helper virus for large-scale preparation of HDAd/HA. The HDAd/HA was obtained and purified twice with CsCI density ultracentrifugation and observed morphologically under transmission electron microscope, and the expression of HA protein was analyzed with RTPCR. Recombinant HDAd/HA expressing HA protein was successfully constructed which could pave the way for in vivo investigation on immunogenicity and efficacy against S-OIV A H1N1 infection.

Recombinant influenza viruses as delivery vectors for hepatis B virus epitopes

PURPOSE: Neuraminidase (NA) of influenza virus contains stalk region that shows a great deal of variability in both amino acid sequence and length. In this paper, we investigated generation of recombinant influenza viruses that had hepatitis B virus (HBV) B cell epitopes in the NA stalk region as a dual vaccine candidate. MATERIALS AND METHODS: We used the WSH-HK reassortant helper virus for rescue of recombinant influenza virus containing HBV epitopes and reverse genetic protocol based on the use of micrococcal nuclease-treated virus cores for reconstitution of ribonucleoproteins. RESULTS: We successfully generated a chimeric influenza viruses which contained 22 amino acid peptides in the stalk region derived from the surface and pre-surface protein HBV. The growth kinetics of the recombinant viruses was investigated after infection of Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney (MDBK) cells and the rIV-BVPreS virus showed higher titer than other viruses in MDCK cells. We also confirmed the presence of HBV epitopes in the chimeric viruses by enzyme-linked immunosorbent assay (ELISA) using anti-HBV polyclonal antibody. When the ratio of recombinant virus verse wild type virus was calculated by ELISA, recombinant viruses exhibited 2 fold higher values than the wild type virus. CONCLUSION: These results suggest that chimeric influenza virus which contained foreign antigens can be used as dual vaccine against both HBV and influenza viruses.

In vitro transgenic expression efficacy of a helper-dependent adenoviral vector encoding enhanced green fluorescent protein / 生物工程学报

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

A novel system for producing lentiviral vectors / 生物工程学报

The aim was to develop a cell culture system capable of producing high titer lentiviral vector stocks with recombinant vaccinia viruses as helpers. BHK21 was co-transfected by three main plasmids containing the transducing plasmid pVECRNA, the packaging plasmid pGAGPOL and the envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamine2000. After 4 days incubation, the culture supernatant of lentiviral vectors was collected and judged by RT-PCR and the Western blot, the results showed that lentiviral vectors were found in the culture supernatant; approximately (11.71 +/- 0.80) x 10(11) copies of lentiviral vector RNA were present per mL of cell culture supernatant, as detected by Real-time PCR; the vector stocks with titers was up to (1.3 +/- 0.18) x 10(8) tu/mL, as detected by flow cytometry , which is one order of magnitude higher than the output of classical manufacture system. These results suggest that the new poxviral/lentiviral hybrid system for efficient lentiviral vector production was initially established. It provides the basis for the future development of industrial application.

Large recombinant protein displayed on filamentous phage surface and its interaction with small molecule / 生物工程学报

Recombinant proteins were expressed as fusions with the phagemid system of pHEN-KM13 and the characteristics and activities of the fusion proteins displayed on the surface of filamentous phagintain the were studiedon abilty. The altered titer of rescued phages from the phagemid system after trypsin treatment indicated the relative quantity of the phages displaying fusion proteins. The rescue phages displayed foreign proteins could keep the bacterial infection ability, while the bald phage without foreign protein displayed on its surface was sensitive to trypsin treatment and lost the bacterial infection ability. To determine the upper limit for filamentous phage display, four recombinant proteins, glutathione-S-transferase and glutathione-S-transferase fused with three various size peptide linkers, were fused to N terminus of capsid protein gp3 and rescued by helper phage KM13. The rescued phages which displayed fused protein with the size of 40kD or less maintain the infection ability. To assay the activity of the phage displayed protein, the known small molecule probe was used in the interaction study with protein incorporated on phage surface. Results showed that the glutathione-S-transferase on phage surface still bound to glutathione specifically. It indicated that the glutathione-S-transferase displayed on phage surface was correctly folded and functionally active. The results demonstrated that it was feasible to use small molecule probes to interact with the protein displayed on phage surface. In turn, the method described here also demonstrated that phage display system could be utilized to investigate the interactions between protein and small molecules.

Rescue of Synthetic Hantavirus-Like Foreign RNA by Hantavirus as a Helper Virus

A system for the expression of synthetic hantavirus-like luciferase RNA was developed using Hantaan (HTN) virus as a helper virus. The hantavirus-like luciferase RNA was constructed by the deletion of the coding region in HTN virus (76~118) S genome and by replacement of a luciferase gene. PCR was performed using primers designed to amplify the whole region of hantavirus-like foreign gene. The resulting PCR product was placed under the control of the T7 RNA polymerase promoter for in vitro transcription. The produced hantavirus-like luciferase RNA was transfected into Vero-E6 cell infected with HTN virus using lipofectin. The level of expression was measured using a luminometer. The hantavirus-like luciferase RNA was allowed to amplify and express. And also, the hantavirus-like luciferase RNA was packaged into HTN virions. The 5' terminal and 3' terminal conserved sequences of HTN virus genome were sufficient to provide the signals for RNA amplification and packaging. This suggests that the RNA promoter region for hantavirus RNA synthesis is located in 3' terminal region. The luciferase activity was analyzed from the progeny virus-infected cells in order to examine if the 5' and 3' terminal sequences play a role in regulating a packaging pathway while genome of HTN virus is packaged. The luciferase activity was detectable in every cell passage. However, the activity of luciferase was decreased gradually after each passage. The fact that the hantavirus-like luciferase RNA can be packaged into progeny virus suggests that the 5' and 3' terminal sequences of HTN virus genome play an important role in regulating a packaging pathway.

Pseudotype HIV-1 Particles Carrying CD4

A defective HIV-1 helper virus DNA, pHyPC, was assembled by deleting the RNA packaging signal, env, nef and the 3'LTR sequences. HIV-1 like virus particles that carry the HIV-1 receptor, CD4 were generated by coexpression of pHyPC and plasmid DNAs encoding different chimeric CD4 proteins. The CD4 particles, sharing the CD4 ectodomain, precisely fused to different membrane anchors. CD4(+) particles specifically bound to HIV-1 Env expressing cells, but any signs of infection into these cells were not detected. Binding was only partially blocked by either polyclonal anti-CD4 antibodies or by high concentrations of soluble CD4. Suprisingly, CD4(+) particles also adsorbed to HeLa, CHO, NIH3T3 and COS-7 cells in the absence of HIV-1 Env expression. Adsorption was comparable in strength and speed to the highly specific CD4-Env interaction. CD4(-) particles exhibited only background levels of binding. Cell binding was CD4- dependent, but it was independent of the cell type from which the CD4(+) particles originated. Interestingly, CD4-dependent/Env-independent binding was only found when CD4 was present on virus particles. This suggests that the micro-environment of CD4 on virus particles uniquely expose this new cell binding activity. Its high affinity could explain in part why infection of Env(+) cells by CD4(+) particles was not detected. Further experiments will be required to evlauate whether this strong membrane interaction could represent one step in the multiple-step viral entry process.

Identification of Retroviral Vectors Producing High Viral Titer

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are. focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer, To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of leo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.

Comparison of Efficiency of Infection of Human Cancer Cell Lines Via Retroviral Vector System / 대한암학회지

PURPOSE: There are several reasons why retroviruses are useful as vectors for gene therapy. However, retroviral vectors also have some limitations. Research in retroviral-mediated gene transfer has struggled with low titer and transduction efficiency on certain human target cells even with the addition of polycations to enhance transduction. Efficient in vivo gene transfer with retroviral vectors will require the availability of large amounts of vector at titers higher than generally possible by most current methods. Therefore, transduction efficiency of various human cell types with retroviral vector system is very important in human gene therapy. In an effort to test the transduction efficiency of a retrovial vector in the human cancer cell lines, a retroviral vector was infected into various human cancer cell lines. MATERIALS AND METHODS: We generated retrovirus producing cell lines through transfection or infection of amphotropic packaging cell line PA317 with ecotropic retroviruses encoding bacterial lacZ gene. The amphotropic retrovirus vector was used to transduce various human cancer cell lines. RESULTS: Of eight randomly chosen G418-resistant clones generated by transfection, only two clone produced the vector at up to >10 (6) cfu/ml, while one of five clones generated by infection yielded higher-titer virus in the absence of helper virus, up to 1 X 10 (7) cfu/ml, than the transfected clones. Transduction with supernatant derived from a PA317 producer cell line has resulted in transduction levels from 1% to 15%, 5- to 60-fold lower than those analyzed in NIH3T3 cells. CONCLUSION: These findings suggest that new improved gene transfer method into human cancer cells using retroviruses is required for efficient in vivo cancer gene therapy.