Adenoviridae ; genetics ; Gene Expression ; Hepatic Stellate Cells ; metabolism ; Humans ; Urokinase-Type Plasminogen Activator ; genetics ; metabolism

OBJECTIVETo observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.

METHODSThe recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.

RESULTSInfection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.

CONCLUSIONThere is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.

Adenoviridae ; genetics ; Centrosome ; metabolism ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; Transfection ; Tubulin ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.

METHODSTotal RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.

RESULTHigh expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.

CONCLUSIONAd/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

Adenoviridae ; genetics ; Catalase ; genetics ; metabolism ; Cell Line ; Genetic Vectors ; Humans ; Transfection

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMax^TM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.
Adenoviridae/genetics* ; Cell Proliferation ; Estrogen Receptor alpha/metabolism* ; Recombinant Proteins ; Transfection

OBJECTIVETo purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro.

METHODSThe recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method.

RESULTSThe recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P < 0.05).

CONCLUSIONThe purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.

Adenoviridae ; Cell Division ; Cells, Cultured ; Glycoproteins ; isolation & purification ; Glycosylation ; Humans ; Recombinant Proteins ; isolation & purification ; metabolism ; Repressor Proteins ; isolation & purification ; metabolism

OBJECTIVETo construct mouse enhanced green fluorecence protein (EGFP) -peroxisome proliferator-activated receptor (PPAR)gamma2, and to detect EGFP-PPARgamma2 expression in infected mouse bone marrow mesenchymal stem cells (BMSC).

METHODSCut the fragment of PPARgamma2 from the expression plasmid pcDNA flag PPARgamma2, then cloned the gene fragment into pEGFP-C1 and pEGFP-N1 vector. Subsequently, subclone the fragment EGFP-PPARgamma2 from pEGFP-C1-PPARgamma2 into the shuttle plasmid DC315. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid DC315-EGFP-PPARgamma2 and large adenovirus helper plasmid pBHGlox deltaE1, 3Cre in mediation of liposome. The obtained replication-defective recombinant adenovirus Ad-EGFP-PPARgamma2 was confirmed. Then it was propagated in HEK293 cells. After the BMSC were transfected for 72 h, adipogenic differentiation was demonstrated.

RESULTSHEK293 cells were transfected with the pEGFP-C1-PPARgamma2 or pEGFP-N1-PPARgamma2 in mediation of liposome. The former green fluorescence protein was better than the latter by fluorescence microscope. The recombinant plasmids were digested and identified. Western blot analysis showed the expression of EGFP-PPARgamma2 in vitro. EGFP-PPARgamma2 protein was detectable in the nucleus of BMSC.

CONCLUSIONThe recombinant adenovirus encoding EGFP-PPARgamma2 fusion protein was successfully constructed, which provided a basis for application of EGFP-PPARgamma2 gene to adenovirus-mediated gene therapy.

Adenoviridae ; Animals ; Bone Marrow Cells ; metabolism ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; HEK293 Cells ; Humans ; Mesenchymal Stromal Cells ; metabolism ; Mice ; PPAR gamma ; metabolism ; Recombinant Proteins ; metabolism ; Transfection

OBJECTIVETo investigate the genetic stability of non-replicating recombinant adenovirus which used Ad41 as vector and could express VP6 gene of group A rotavirus during continous passage, in order to develop the vaccine of rotavirus.

METHODSThe recombinant adenovirus rvAd41-VP6 (o) was prepared by our laboratory early, it then was continuously propagated on 293TE7 cells for 14 passages. After that samples of the infected cells were collected at every 2 passages for the detection of the integration of the VP6 gene by PCR, and the expression of the target protein was detected by Western Blot analysis.

RESULTSAnalysis by PCR revealed that, there was stable integration of specific VP6 gene in the rvAd41-VP6 (o), Western Blot analysis confirmed that rvAd41-VP6 (o) could stably expressed the group-specific antigen structural protein VP6 (o), and it had preferable genetic stability.

CONCLUSIONThe recombinant adenovirus rvAd41-VP6 (o) which could stably express the VP6 (o) gene had favorable biological property in vitro, and it has provided a basis for further research of animal immunization.

Adenoviridae ; genetics ; metabolism ; Antigens, Viral ; genetics ; metabolism ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Humans ; Rotavirus ; genetics ; metabolism ; Rotavirus Infections ; virology

OBJECTIVEA strain of replication deficient recombinant adenvirus encoding fusion glycoprotein (F) of subgroup A human respiratory syncytial virus (RSV) was constructed and the expression of F was identified.

METHODSThe F gene was obtained from pGEM3zf-F with Xho I and Hind III, cloned into adenoviruse shuttle vector pShuttle-CMV,and then the resulting pShuttle-CMV/F was transformed into E. coli BJ5183/p with pAdeasy-1 to produce pre-adenoviral plasmid encoding F by homologous recombination. This resultant plasmid was linearized by digestion with Pac I and transfected into 293 packaging cells to generate FGAd-F. Finally, the expression of F protein was identified by Western Blot analysis.

RESULTSFGAd/F was successfully constructed, and the expression of RSV F protein was identified by Western Blot.

CONCLUSIONWe have obtained a strain of replication-defective adenovirus FGAd/F encoding RSV F protein, which can be used further to investigate its protective efficacy against RSV infection in vivo.

Adenoviridae ; genetics ; Adenoviridae Infections ; genetics ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; genetics ; metabolism ; Respiratory Syncytial Virus, Human ; genetics ; Respiratory Syncytial Viruses ; genetics ; Viral Fusion Proteins ; genetics ; metabolism ; Virus Replication

The present study is aimed to study the effect of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) gene transfer on the contractile function of isolated cardiomyocytes of canines. The cardiomyocytes were isolated with collagenases. The isolated cardiac cells were divided into untransfected group, empty vector group and SERCA2a-transfected group. Recombinant adenovirus vector carrying enhanced green fluorescent protein gene was used for SERCA2a gene delivery. The expression of SERCA2a protein in cardiomyocytes was determined by Western blot. Contractile function of cardiomyocytes was measured with motion edge-detection system of single cell at 48 h after transfection. The results showed, compared with untransfected group, SERCA2a protein level, percentage of peak contraction amplitude under normal condition, percentages of peak contraction amplitude under Ca(2+) or isoproterenol stimulation, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) in SERCA2a-transfected group all increased significantly. While all the above indices in empty vector group did not show any differences with those in untransfected group. These results suggest that the overexpression of SERCA2a by gene transfer may enhance the contraction function of canine myocardial cells.
Adenoviridae ; genetics ; metabolism ; Animals ; Dogs ; Male ; Myocardial Contraction ; drug effects ; physiology ; Myocytes, Cardiac ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism ; Transfection

OBJECTIVETo construct a recombinant adenovirus carrying human endostatin gene with AdEasy system.

METHODSEndostatin gene fragment was amplified from Pshuttle-Endostatin plasmid with PCR and subcloned into the pAdTrack-CMV shuttle vector. The resultant plasimid was cotransduced into E.coli BJ 5183 cells with pAdEasy-1 plasmid for homologous recombination. The linearized recombinant plasmid was subsequently transfected into AAV 293 cells, and the recombinant adenovirus was detected by GFP, PCR and restriction analysis.

RESULTS AND CONCLUSIONThe positive clones of the recombinants were verified by restriction analysis and the titer of the virus reached 2.06 x 10(10)pfu/ml, suggesting successful construction of recombinant adenovirus pAd-Endo.

Adenoviridae ; genetics ; metabolism ; Cell Line ; Cloning, Molecular ; Endostatins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Humans