Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

CONSTRUCTION OF THE DICISTRONIC ADENOVIRUS VECTOR EXPRESSING BIOACTIVE HUMAN INTERLEUKIN-12

The full-length cDNA encoding the subunits p40 and p35 of human interleukin12(hIL12) were cloned separately by RTPCR, linked together by internal ribosomal entry site (IRES) of encephalomyocarditis virus which initiates capindependent translation to form a dicistronic gene fragment. The dicistronic fragment was placed between the cytomegalovirus (CMV) promoter and SV40 polyA signal to form a dicistronic expression cassette. Subsequently, the dicistronic expression cassette was inserted into E1 region of Ad5 genome in cosmid vector pAx1cw of E1substitution type. By homologous recombination with EcoT22Idigested Ad5 DNATPC in 293 cells, the replicationdeficient recombinant adenoviruses of hIL12 were generated efficiently. After infected with hIL12 recombinant adenoviruses in vitro, 293 cells, human hepatocellular carcinoma cells HepG2, and primary human skin fibroblasts expressed and secreted hIL12 at comparable levels (30～60ng/ 106cells/24hr), which could stimulate the proliferation and IFNγ production of human lymphoblasts. These suggest that the dicistronic adenovirus vector of hIL12 could effectively mediate the expression of bioactive hIL12 and might be used in cancer gene therapy.

Recombinant adenovirus vector Ad-hIL-10 protects grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats

BACKGROUND: Interleukin 10 (IL-10), a Th2 type cytokine, modulates inflammatory responses by inhibiting the production of proinflammatory cytokines. This study was designed to investigate the protective effects of adenovirus-mediated human IL-10 (Ad-hIL-10) gene transfer on protecting grafts from cold ischemia-reperfusion injury following orthotopic liver transplantation in rats. METHODS: Adenoviruses encoding hIL-10 or beta-galactosidase (Ad-lacZ) were injected via the superior mesenteric vein into prospective donor animals. The donor liver was harvested 48 hours after transduction, and stored for 12 hours at 4 degrees C in lactated Ringer's solution prior to transplantation. The rats were divided into saline, Ad-lacZ, and Ad-hIL-10 groups. Liver function test, histopathological examination, reverse transcriptase-polymerase chain reaction (RT-PCR), and Western blotting were performed at 24 hours after transplantation in the three groups. RESULTS: Liver function (ALT and AST) was significantly improved, and the Suzuki score was significantly decreased in the Ad-hIL-10 group. The levels of hepatic TNF-alpha, MIP-2, ICAM-1 mRNA, and NF-kappa B protein in the Ad-hIL-10 group were significantly decreased. The expression of hIL-10 mRNA was detected by RT-PCR in Ad-hIL-10-treated grafts but not in controls treated with saline or Ad-lacZ. CONCLUSIONS: Donor pretreatment with Ad-hIL-10 down-regulates the expression of proinflammatory cytokines TNF-alpha, MIP-2, and ICAM-1 mRNA. hIL-10 protects against hepatic cold ischemia-reperfusion injury, at least in part, by suppressing NF-kappa B activation and subsequent expression of proinflammatory mediators. (Hepatobiliary Pancreat Dis Int 2010; 9: 144-148)

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer

BACKGROUND:Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants,but has potential for systemic immune inhibition.This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation.METHODS:A normal human liver cell line(L02) was transfected with adenovirus vector containing the CTLA4Ig gene(Ad-CTLA4Ig-EGFP) in vitro,and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining.Transfected cells then were injected into the spleen of Sprague-Dawley rats,the survival of cells was determined by immunohistochemistry,and the immune status was examined through CD4 + and CD69 + T cellcounts and ELISA detection of IL-2 in peripheral blood.RESULTS:L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks.Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation,while none was detected in the control group.Furthermore,the percentages of CD4 + and CD4 + CD69 + T cells in the CTLA4-transfected group were 24.5% and 45.1%,markedly lower than those in the control group at 4 weeks post-transplantation(P<0.01).Furthermore,the IL-2 level was also lower in the CTLA4transfected group than in the control group.CONCLUSION:Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

In situ tumor vaccination with adenovirus vectors encoding measles virus fusogenic membrane proteins and cytokines

AIM： To evaluate whether intratumoral expression of measles virus fusogenic membrane glycoproteins H and F （MV-FMG）, encoded by an adenovirus vector Ad.MV-HI F, alone or in combination with local coexpression of cytokines （IL-2, IL-12, IL-18, IL-21 or GM-CSF）, can serve as a platform for inducing tumor-specific immune responses in colon cancer.METHODS： We used confocal laser scanning microscopy and flow cytometry to analyze cell-cell fusion after expression of MV-FMG by dye colocalization. In a syngeneic bilateral subcutaneous MC38 and Colon26 colon cancer model in C57BL/6 and BALB/c mice, we assessed the effect on both the directly vector-treated tumor as well as the contralateral, not directly vector- treated tumor. We assessed the induction of a tumorspecific cytotoxic T lymphocyte （CTL） response with a lactate dehydrogenase （LDH） release assay.RESULTS： We demonstrated in vitro that transduction of MC38 and Colon26 cells with Ad.MV-H/F resulted in dye colocalization, indicative of cell-cell fusion, in addition, in the syngeneic bilateral tumor model we demonstrated a significant regression of the directly vector-inoculated tumor upon intratumoral expression of MV-FMG alone or in combination with the tested cytokines. We observed the highest anti-neoplastic efficacy with MV-FMG and lL-21 coexpression. The degree of tumor regression of the not directly vector-treated tumor correlated with the anti-neoplastic response of the directly vector-treated tumor. This regression was mediated by a tumor-specific CTL response.CONCLUSION： Our data indicate that intratumoral expression of measles virus fusogenic membrane glycoproteins is a promising tool both for direct tumor treatment as well as for tumor vaccination approaches that can be further enhanced by cytokine coexpression.

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.

Construction and characterization of calreticulin-HBsAg fusion gene recombinant adenovirus expression vector

AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.

Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rht, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG- FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment （3.97 kb） obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect （CPE） appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A a （Ppp3ca） cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

Gene therapy for allergic rhinitis with recombinant adenovirus vector containing CTLA4Ig in mice

Objective: To examine the role of recombinant adenovirus vector containing CTLA4Ig gene(Ad-CTLA4Ig) in the treatment of induced allergic rhinitis in mice.Methods: Allergic rhinitis was induced by sensitizing and challenging with ovalbumin(OVA).Ad-CTLA4Ig was intraperitoneally injected 30 min before OVA challenge.Adenovirus vector without inserted CTLA4Ig cDNA served as the control.The symptoms and morphological changes of nasal mucosa of each group were observed, and the serum levels of IgE against OVA were detected with ELISA.Results: There were no obvious symptoms and pathological changes in Ad-CTLA4Ig treated group, in which the serum OVA-specific IgE levels were significantly lower than that in control groups(P< 0.05).Conclusion: Ad-CTLA4Ig prevents and treats allergic rhinitis of mice,implying the possibility of the usage of Ad-CTLA4Ig against allergic rhinitis in clinic in future.

Construction and Expression of Human PTEN Tumor Suppressor Gene Recombinant Adenovirus Vector

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad- PTEN containing green fluorescent protein （GFP） was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 × 10^10 pfu/mL, and about 70 % breast cancer cells were infected with Ad-PTEN when multiplicity of infection （MOI） reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

THE ROLE OF RECOMBINANT Rb GENE ADENOVIRUS VECTOR IN THE GROWTH OF LUNG ADENOCARCINOMA CELLS

Objective: To study the role of the most extensively studied tumor suppressor gene, retinoblastoma (Rb) gene, on the growth of lung adenocarcinoma cell line GLC 82 and explore a gene therapy approach for lung adenocarcinoma Methods: The recombinant Rb gene adenovirus vector was constructed, the control virus which carries LacZ gene was producted by the same method Infection effects were detected by biochemical staining of β gal and immunohistochemical analysis of Rb protein The Rb cDNA of infected cells were determined by PCR The cell growth rate and cell cycle were observed by cell counting and flow cytometry Results: The constructed recombinant adenovirus vector could infect effectively the cells with high level expression of Rb cDNA and Rb protein The transfection of wild type Rb gene could suppress GLC 82 cell proliferation and decrease the cellular DNA synthesis Conclusions: These results showed the possibility of using recombinant Rb gene adenovirus vector in the gene therapy of cancer to inhibit the growth of cancer

CONSTRUCTION OF A RECOMBINANT ADENOVIRUS VECTOR OF HUMAN PAPILLOMAVIRUS TYPE 16 L1_E7C

Objective: Human papillomaviruses are closely associated with human cervical cancer, especially HPV types 16 and 18. At present, HPV can not be produced in large quantity; it also has tumorgenicity and these properties of HPV have seriously hampered the development of HPV vaccine. HPV type 16 LI proteins can assembled into virus-like particles (VLP), which are morphologically identical to the nature virion. In order to develop the recombinant adenovirus vectors of HPV, we constructed a recombinant adenovirus shuttle plasmid pCA14 L1-E7C. Methods: Human papillomavirus type 16 LI open reading frame without terminator codon (TAA) (5559–7152) and E7C (682–855) were amplified using PCR. The L1 and E7C fragments were inserted into pGEM-T easy vectors by T-A strategy, named pTAL1 and pTAE7C. pTAL1 was cut with Hind III and BgIII, the pTAE7C with BamHI and ClaI. The L1 DNA fragment, E7C and pBluesscript SK were ligated together using T4 DNA ligase. pBSL1-E7C and pBSL1-E7C was digested with Hind III and Xhol. The L1-E7C fragment was inserted into adenovirus shuttle plasmids pCA14, named pCA14L1-E7C. DNA sequence results indicated that The L1-E7C DNA fragment can encode the HPV16L1-E7 fusion protein correctly. Results: The L1 and E7C DNA fragments were amplified by PCR and recombinant plasmid pTAL1, pTAE7C, pBSL1-E7C and pCA14L1-E7C were constructed correctly. The pCA14L1-E7C can be used in the further research work, cotransfected the 293 cell with the parent adenovirus pBHG10. Conclusion: Our results indicated that we have constructed a HPV16L1-E7 fusion DNA fragments and the adenovirus shuttle plasmids pCAL1-E7C for the further research.

Preparation and identification of monoclonal antibodies against the adenovirus vector

Objective： To prepare and identity monoclonal antibodies （McAbs） against the capsid proteins of adenovirus vector. Methods： BALB/c mice were immunized with a mixture of the purified adenovirus vector （Adv） and AI（OH）3. McAbs were produced using cell fusion technique in a conventional way. The sensitivity and specificity of monoclonal antibodies was identified by indirect enzyme linked immunosorbent assay （ELISA）, immunocytochemical staining and Western blotting. Results： Six strains of hybridoma cells （A4H11, A8C7, F1H5, G1D2, G4E3 and H2G8） that can stably secrete the IgG1 McAb against Adv were obtained. After 3 months subculture and low concentration of serum adapting culture, six strains retained their stability to secrete McAb. The ascites titers were between 1：10^6 and 1：10^8. Western blot analysis demonstrated that all the McAbs reacted with one protein （about 114 kDa） which is present in wild type 3 adenovirus （wtAd3）, wild type 5 adenovirus （wtAd5）, wild type 7 adenovirus （wtAd7） and adenovirus vector. Conclusion： Successfully prepared six strains of hybridoma cell secreted monoclonal antibodies against the hexon proteins of adenovirus vector, and provided the substantial foundation of preclinical research of adenovirus vectors.

A HEVC Video Steganalysis Method Using the Optimality of Motion Vector Prediction

Among steganalysis techniques,detection against MV(motion vector)domain-based video steganography in the HEVC(High Efficiency Video Coding)standard remains a challenging issue.For the purpose of improving the detection performance,this paper proposes a steganalysis method that can perfectly detectMV-based steganography in HEVC.Firstly,we define the local optimality of MVP(Motion Vector Prediction)based on the technology of AMVP(Advanced Motion Vector Prediction).Secondly,we analyze that in HEVC video,message embedding either usingMVP index orMVD(Motion Vector Difference)may destroy the above optimality of MVP.And then,we define the optimal rate of MVP as a steganalysis feature.Finally,we conduct steganalysis detection experiments on two general datasets for three popular steganographymethods and compare the performance with four state-ofthe-art steganalysis methods.The experimental results demonstrate the effectiveness of the proposed feature set.Furthermore,our method stands out for its practical applicability,requiring no model training and exhibiting low computational complexity,making it a viable solution for real-world scenarios.

Whole Perfect Vectors and Fermat’s Last Theorem

A naïve discussion of Fermat’s last theorem conundrum is described. The present theorem’s proof is grounded on the well-known properties of sums of powers of the sine and cosine functions, the Minkowski norm definition, and some vector-specific structures.

Experimental Study of Adenovirus Vector Mediated-hVEGF_(165) Gene on Prevention of Restenosis after Angioplasty

This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF 165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF 165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF 165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (IT max and MT max) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-l were recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

Construction and Identification of Recombinant Adenovirus Vector Containing the hVEGF_(165) Gene

To construct the recombinant adenovirus vector containing the cDNA for human vascular endothelial growth factor (hVEGF 165 ), the cDNA for hVEGF 165 was subcloned into pACCMV·pLpA. Subsequently, this recombinant pACCMV·hVEGF was co transfected into 293 cells together with pJM17 to obtain the replication deficient recombinant adenovirus containing hVEGF gene-AdCMV·hVEGF. The VEGF gene expression was detected by using RT PCR and Western blot in rabbit aorta vascular smooth muscle cells (VSMC) infected with AdCMV· hVEGF. Cultured human umbilical vein endothelial cells (HUVEC) were incubated with the conditioned medium (CM) from above mentioned VSMC infected with AdCMV·hVEGF to observe the effect of VEGF on proliferation of HUVEC. 48 h after the infection with AdCMV·hVEGF, VSMC demonstrated VEGF expression, and the expressed VEGF could stimulate the proliferation of HUVEC in vitro . Successfully prepared AdCMV·hVEGF 165 could express biologically active VEGF in infected VSMC, and stimulate proliferation of HUVEC.

Use of adenovirus vector expressing the mouse full estrogen receptor alpha gene to infect mouse primary neurons

Estrogen plays important regulatory and protective roles in the central nervous system through estrogen receptor a mediation. Previous studies applied eukaryotic expression and lentiviral vectors carrying estrogen receptor a to cladfy the underlying mechanisms. In the present study, an adenovirus vector expressing the mouse full estrogen receptor a gene was constructed to identify biological characteristics of estrogen receptor a recombinant adenovirus infecting nerve cells. Primary cultured mouse nerve cells were first infected with estrogen receptor a recombinant adenovirus at various multiplicities of infection, followed by 100 multiplicity of infection. Results showed overexpression of estrogen receptor α mRNA and protein in the infected nerve cells. Estrogen receptor a recombinant adenovirus at 100 multiplicity of infection successfully infected neurons and upregulated estrogen receptor a mRNA and protein expression.