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

OBJECTIVETo increase the recombinant adenovirus vector mediated human rotavirus G1VP7 and G3VP7 gene expression through coden optimization.

METHODSWe have artificially synthesized two genes of group A human rotavirus that encode G1VP7 and G3VP7 according to the human biased codon. The modified genes were transfected into 293 cells using adenovirus vectors and the gene products, the respective proteins were produced. The expression level of optimized genes and wild type genes was detected by Western Blot.

RESULTSA remarkable increase of the expression level of optimized G1VP7 and G3VP7 genes in comparison with the wild type control.

CONCLUSIONThe coden optimization indeed help increasing the recombinant adenovirus mediated human rotavirus gene expression, which indicated the potential applicationof such recombinant adenoviruses in the development of adenoviral-vectored rotavirus vaccines.

Adenoviridae ; genetics ; Antigens, Viral ; genetics ; metabolism ; Cloning, Molecular ; methods ; Codon ; genetics ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Rotavirus ; genetics ; 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

Human Sodium/Iodide symporter gene cDNA was amplified from thyroid tissue of the patient suffering from Graves disease by RT-PCR, and T/A cloned into pGEM-TEasy-NIS for sequencing, subcloned into shuttle plasmid pAdTrack-CMV which contained a green fluorescent protein (GFP) gene, and then forwarded to homologous recombinant in the bacteria BJ5183 that already contained AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the 293 cells to obtain recombinant adenovirus. The results showed that the recombinant AdNIS was correctly constructed and confirmed by restriction enzyme analysis and PCR. The viral titer was 2. 5 - 3 x 10(9) efu/ml. So, the recombinant adenovirus vector carrying hNIS was successfully constructed, thus providing a basis for researches on 131I therapy in nonthyroid carcinoma.
Adenoviridae ; genetics ; metabolism ; DNA, Complementary ; genetics ; Genetic Vectors ; genetics ; metabolism ; Graves Disease ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Symporters ; biosynthesis ; genetics

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

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

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.
Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Helper Viruses ; genetics ; metabolism ; Humans ; Transgenes ; Viral Fusion Proteins ; genetics ; metabolism

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.
Adenoviridae ; genetics ; metabolism ; Defective Viruses ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Monocytes ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection ; U937 Cells

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