Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

Hepatitis B virus envelope as a targeting gene transfer vector for hepatic cancer cells

Objective： The aim of the study was to observe the transfection efficacy of hepatitis B virus envelope （HBVE） and evaluate its ability as a gene transfer vector for liver cancer cells. Methods： To obtain HBVE, the supematant fluid of HepG 2.2.15 cells was mixed with a PEG8000 solution for concentration and was inactivated by β-propiolactone. The acquired HBVE was used to pack plRES2-EGFP to test its package ability. Then, we examined its quantity and quality with ELISA, PCR, SDS-PAGE and electron microscopy. The plRES2-EGFP was packed with HBVE and obtained the product HBVE-GFP. The plRES2-EGFP was packed with liposome and obtained the product liposome-GFP. HBVE-GFP and liposome-GFP were used to transfer HepG 2 cells to study the transfection efficiency. HBVE-GFP was used to transfer HepG 2, A549, HeLa and FB cells to study the targeting ability. The green fluorescent protein （GFP） expression was observed under a fluorescent microscope. The rate of GFP positive cells was determined by flow cytometry. Results： 1. The acquired HBVE could retain the surface protein HBsAg ＋ pre S1 ＋ pre S2 and had no virus DNA. It had good package ability for plRES2-EGFP. 2. Transfection efficiency： The GFP could be observed in both the liposome group and HBVE group under the fluorescent microscope. But the HBVE group had a higher fluorescent intensity than liposome group. The transfection rate of liposome group was 49.97% ＋ 2.37% while the HBVE group was 70.65% ＋ 3.15% and the fluorescent intensity of the HBVE group was 3-4 times （P = 0.000） for liposome group with the determination of flow cytometry. 3. Targeting ability： The GFP could be observed in the four groups under the fluorescent microscope. The HepG 2 group had the highest fluorescent intensity among the four groups. The transfection rate of HepG 2 group was 71.35% ＋ 0.03% which was highly expressed than other groups （P = 0.000） and the fluorescent intensity of the HepG 2 group was 2-3 times （P = 0.000） for the other 3 groups with the determination of flow cytometry. Conclusion： HBVE can be constructed successfully with the methods of PEG8000 and β-propiolactone from the supernatant fluid of HepG 22.15 cells. The HBVE can be a candidate gene transfer vector for liver cancer cells.

An Improved Atmospheric Vector Radiative Transfer Model Incorporating Rough Ocean Boundaries

The radiative transfer model (RT3), a vector radiative transfer (VRT) scheme in a plane-parallel atmosphere, was bounded by a rough ocean surface in this study. The boundary problem was solved using a Fourier series decomposition of the radiation field as a function of the azimuth. For the case of a rough ocean surface, the decomposition was obtained by developing both the Fresnel reflection matrix and the probability distribution of the water facet orientation as Fourier series. The effect of shadowing by ocean surface waves was also considered in the boundary condition. The VRT model can compute the intensity and degree of polarization of the light at the top of the atmosphere (TOA), the ocean surface, and any level of the atmosphere in the ocean-atmosphere system. The results obtained by our model are in good agreement with those computed by Ahmad’s model. The simulated results showed that the shadow effects of wave facets on the intensity and the degree of polarization are negligible except at the ocean surface near the grazing angle, possibly because we did not consider the effect of white caps.

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.

Multi-coupled single scattering method of solving vector radiative transfer equations

A new method of multi-coupled single scattering （MCSS） for solving a vector radiative transfer equation is de- veloped and made public on Internet. Recent solutions from Chandrasekhar＇s X-Y method is used to validate the MCSS＇s result, which shows high precision. The MCSS method is theoretically simple and clear, so it can be easily and credibly extended to the simulation of aerosol/cloud atmosphere＇s radiative properties, which provides effective support for research into polarized remote sensing.

Vector Radiative Transfer in a Vertically Inhomogeneous Scattering and Emitting Atmosphere.PartⅠ:A New Discrete Ordinate Method

The original vector discrete ordinate radiative transfer(VDISORT)model takes into account Stokes radiance vector but derives its solution assuming azimuthal symmetric surface reflective matrix and atmospheric scattering phase matrix,such as the phase matrix derived from spherical particles or randomly oriented non-spherical particles.In this study,a new VDISORT is developed for general atmospheric scattering and boundary conditions.Stokes vector is decomposed into both sinusoidal and cosinusoidal harmonic modes,and the radiance at arbitrary viewing geometry is solved directly by adding two zero-weighted points in the Gaussian quadrature scheme.The complex eigenvalues in homogeneous solutions are also taken into full consideration.The accuracy of VDISORT model is comprehensively validated by four cases:Rayleigh scattering case,the spherical particle scattering case with the Legendre expansion coefficients of 0th-13th orders of the phase matrix(hereinafter L13),L13 with a polarized source,and the randomoriented oblate particle scattering case with the Legendre expansion coefficients of 0th-11th orders of the phase matrix(hereinafter L11).In all cases,the simulated radiances agree well with the benchmarks,with absolute biases less than 0.0065,0.0006,and 0.0008 for Rayleigh,unpolarized L13,and L11,respectively.Since a polarized bidirectional reflection distribution function(pBRDF)matrix is used as the lower boundary condition,VDISORT is now able to handle fully coupled atmospheric and surface polarimetric radiative transfer processes.

Vector Radiative Transfer in a Vertically Inhomogeneous Scattering and Emitting Atmosphere. Part Ⅱ: Coupling with Azimuthally Asymmetric Polarized Bidirectional Reflection over Oceans

The reflection of ocean surface is often assumed azimuthally symmetric in the previous vector discrete ordinate radiative transfer(VDISORT)and many other radiative transfer solvers.This assumption can lead to obvious errors in the simulated radiances.In this study,the vector radiative transfer equation is solved with a polarized bidirectional reflection distribution function(pBRDF)for computing the surface-leaving radiation from the lower boundary.An azimuthally asymmetric pBRDF model at visible and infrared bands over oceans is fully coupled with the updated VDISORT model.The radiance at the ocean surface is combined with the contributions of atmospheric scattering and surface properties.It is shown that the radiance at the ocean surface also exhibits a strong angular dependence in the Stokes vector and the magnitudes of I.Q.and V increase for a larger azimuthal dependence of pBRDF.In addition,the solar position affects the peaks of sun glitter pattern,thus modulating the signal magnitudes and the angular distributions.As ocean wind increases,the reflection weakens with reduced magnitudes of Stokes parameters and lessvarying angular distributions.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Research on Simulation and Prediction of Internal Combustion Engine Structural Acoustic Radiation

With the purpose of efficiently predicting structural radiated noise of internal combustion engine（I.C.E.）,a new simulation technique is introduced,which is an approach based on boundary element method （BEM）,acoustic transfer vector（ATV） technique and coupled boundary element model and finite element model （BEM-FEM） approach.Analyses of vibration exciting loads,computing structural dynamic characteristics and dynamic responses have led to theoretical results,which are tested on an L6 diesel engine to validate this proposed technique in engineering practice.

SEQUENCE STUDY AND RETROVIRUS MEDIATED GENE TRANSFER IN MAMMALIAN CELL SYSTEMS OF HUMAN INTERLEUKIN 2 cDNA

Interleukin 2 (IL- 2) is a T cell growth factor. In the present study, human IL- 2 cDNA was cloned from total RNA of activated tonsillar mononuclear cells (TMNC) following reverse transcription and PCR amplification using a pair of synthesized primers. DNA sequence analysis demonstrated that the IL- 2 cDNA cloned from the Chinese tonsil donor is identical with the data reported so far, reflecting the high structural conservation of this gene. The human IL- 2 cDNA was inserted into XM6 to construct a recombinant retroviral expression vector XM6-IL2, having human IL-2 cDNA driven by the 5' LTR of MMLV. This vector was successfully passaged through ψ 2 and PA317 cells to yield high producer lines of ecotropic and amphotroplc infectious viruses. The murine myeloma cell line SP2/0 after being infected by retrovirus released from high titer PA317 constitutlvely secreted IL- 2 activity into the culture medium when assayed for T cell proliferative capacity. Activated human T cells are also exposed to the infectious retrovirus XM6- IL2. In sharp contrast to the parallel controls, the infected T cells with or without the furtheraddition of 500 units/ ml of exogenous IL- 2 proliferated and formed colonies of significant size under the selection pressure of G418. However, their growth in vitro could only be maintained for about 3 weeks. These facts demonstrated that gene transfer of human IL- 2 cDNA viaretrovirus is far from being sufficient to maintain the relatively long-termgrowth and clonal expansion of human T cell subpopulations in vitro.

RETROVIRUS-MEDIATED TNF-α GENE TRANSFER INTO TCA8113 CELLS

Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.

Gene transfer and expression in rat anastomotic artery in vivo using adenoviral vector

Objective To observe the efficiency and time course of gene expression and the safety of adenoviral vector mediated gene transfer in vivo.Methods After soaking soluble stents in a high concentration of glucose solution containing Adv5-CMV (cytomegalovirus) (control group) or Adv5-CMV/LacZ (treatment group) for 30 minutes, the stents were inserted into the lumina of cut rat carotid arteries and end-to-end anastomoses of the cut carotid were performed with standard microvascular surgical techniques. On days 2, 7, 14, 28, 60 and 90 after gene transfer, anastomotic arteries of the two groups were observed. On days 7 and 14, the ascending aortas, hearts, brains, livers, lungs, spleens and kidneys of the treatment group were observed. All samples were analyzed for the presence of β-galactosidase activity and histochemical staining.Results β-galactosidase activity was not detected in the carotid arteries of the control group and organs not directly exposed to adenoviral vector of the treatment group. The amount of β-galactosidase activity (×10-3?U/g tissue) in the treatment group on the 2nd, 7th, 14th, 28th, 60th and 90th day after gene transfer was 3.87, 11.38, 9.8, 6.43, 3.18 and 2.43, respectively. Microscopic examination of sections from vessels of the control group and from the aortas, hearts, brains, livers, lungs, spleens or kidneys of the treatment group revealed no X-gal staining. Microscopic examination of carotid arteries of the treatment group revealed blue-staining in all anastomotic arteries and in all layers of the arterial wall observed on days 7 and 14 after gene transfer.Conclusion Adenoviral vector can effectively infect blood vessels in vivo. After adenoviral vector mediated direct gene transfer into anastomotic rat carotid arteries, recombinant gene expression began on day 2, peaked between days 7 and 14, prominently declined after day 28, and persisted at low levels more than three months. A recombinant gene could be delivered to a specific site by direct gene transfer in vivo by adenoviral vector infection.

Jacobian matrix for near-infrared remote sensing based on vector radiative transfer model

Due to the polarization effects of the Earth’s surface reflection and atmospheric particles’scattering,high-precision retrieval of atmospheric parameters from near-infrared satellite data requires accurate vector atmospheric radiative transfer simulations.This paper presents a near-infrared vector radiative transfer model based on the doubling and adding method.This new model utilizes approximate calculations of the atmospheric transmittance,reflection,and solar scattering radiance for a finitely thin atmospheric layer.To verify its accuracy,the results for four typical scenarios(single molecular layer with Rayleigh scattering,single aerosol layer scattering,multi-layer Rayleigh scattering,and true atmospheric with multi-layer molecular absorption,Rayleigh and aerosol scattering)were compared with benchmarks from a well-known model.The comparison revealed an excellent agreement between the results and the reference data,with accuracy within a few thousandths.Besides,to fulfill the retrieval algorithm,a numerical differentiation-based Jacobian calculation method is developed for the atmospheric and surface parameters.This is coupled with the adding and doubling process for the radiative transfer calculation.The Jacobian matrix produced by the new algorithm is evaluated by comparison with that from the perturbation method.The relative Jacobian matrix deviations between the two methods are within a few thousandths for carbon dioxide and less than 1.0×10-3%for aerosol optical depth.The two methods are consistent for surface albedo,with a deviation below 2.03×10-4%.All validation results suggest that the accuracy of the proposed radiative transfer model is suitable for inversion applications.This model exhibits the potential for simulating near-infrared measurements of greenhouse gas monitoring instruments.

Tangent-impulse transfer from elliptic orbit to an excess velocity vector

The two-body orbital transfer problem from an elliptic parking orbit to an excess veloc-ity vector with the tangent impulse is studied. The direction of the impulse is constrained to be aligned with the velocity vector, then speed changes are enough to nullify the relative velocity. First, if one tangent impulse is used, the transfer orbit is obtained by solving a single-variable function about the true anomaly of the initial orbit. For the initial circular orbit, the closed-form solution is derived. For the initial elliptic orbit, the discontinuous point is solved, then the initial true anomaly is obtained by a numerical iterative approach; moreover, an alternative method is proposed to avoid the singularity. There is only one solution for one-tangent-impulse escape trajectory. Then, based on the one-tangent-impulse solution, the minimum-energy multi-tangent-impulse escape trajectory is obtained by a numerical optimization algorithm, e.g., the genetic method. Finally, several examples are provided to validate the proposed method. The numerical results show that the minimum-energy multi-tangent-impulse escape trajectory is the same as the one-tangent-impulse trajectory.

Effect of human hepatocyte growth factor on promoting wound healing and preventing scar formation by adenovirus-mediated gene transfer

Objective To evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer Methods A total of 12 female New Zealand rabbits were used in this study Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad HGF group 1, Ad HGF group 2, Ad HGF group 3, Ad GFP (a reporter gene) group and the solvent group Immediately after surgery, 6×10 7 pfu Ad HGF, 6×10 8 pfu Ad HGF, 6×10 9 pfu of Ad HGF, 6×10 9 pfu of Ad GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad GFP (6×10 9 pfu) into its wounds Ice slides of wounds from this animal were observed under fluorescence microscopy Another additional rabbit was used to evaluate the expression of HGF and TGFβ1 after transferring Ad HGF (6×10 9 pfu) into each of its wound Immunohistochemistry was used for detection Results The effect of HGF on reducing excessive dermal scarring was observed by adenovirus mediated gene transfer Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over expression of TGFβ1, which plays an essential role in the progression of dermal fibrogenesis Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring Conclusion HGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing