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【Objective】 Annexin A2 (annexin A2, Anxa2) has been reported to regulate bioactivity in various tumors cells. The purpose of this study was to investigate the correlation between the expression of Anxa2 protein and the proliferation and migration abilities of bladder cancer pumc-91 cells. 【Methods】 The ANXA2 sequence was amplified and inserted into the pcDNA3.1(+) vector in order to prepare the pcDNA3.1(+)-ANXA2 plasmid. PcDNA3.1 (+)-ANXA2 was transiently transfected into pumc-91 bladder cancer cells by lipofectamine 2000. Western blotting assay was performed to detect the expression of Anxa2 protein in the blank group, the control group transfected with pcDNA3.1(+), and the experimental group transfected with pcDNA3.1(+)-ANXA2 plasmid. The proliferation ability of pumc-91 cells was detected using Cell Counting Kit-8(CCK8), and the migration level of pumc-91 cells was detected by transwell assay. Differences in detection data among the groups were compared using one-way ANOVA or repeated measures ANOVA. 【Results】 The plasmid construction was successful and the sequencing was absolutely correct. Western blotting assay showed elevated Anxa2 protein expression level in the experimental group compared to the blank and control groups. CCK8 assay suggested that the number of proliferating pumc-91 cells was significantly higher in the experimental group than in the blank group (P<0.001) and the control group (P=0.001). Transwell assay also showed that the number of pumc-91 cells crossing the membrane was significantly higher in the experimental group than in the blank group (P=0.011) and the control group (P=0.027). 【Conclusion】 Our findings suggested that up-expression of Anxa2 may play a critical role in regulating proliferation and migration of bladder cancer pumc-91 cells.
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Objective To compare the transfection efficiency between different transfection methods in human HepG2 and SGC7901/ADM cells so as to provide experimental basis for further study .Methods To electrons fect the enhanced GFP plasmid into HepG2 and SGC7901/ADM cells by lipofection and electroporation methods ,respectively .The survival rates and transfection efficiency were analyzed .Results The efficiency of eGFP vector transfected into HepG2 cells by lipofection was (23 .8 ± 2 .1)% , compared with lipofection method ,the efficiency of eGFP plasmid transfected by electroporation was up to (49 .6 ± 2 .5)% ,and the difference was statistically significant(P<0 .05) .The efficiency of SGC7901/ADM cells by lipofection was (25 .4 ± 1 .3)% ,com‐pared with lipofection method ,the efficiency of electroporation was up to(52 .6 ± 2 .1)% ,and the difference was statistically signifi‐cant(P<0 .05) .This study provides reliable test parameters for electransfection of HepG2 and SGC7901/ADM cells .Conclusion The transfection efficiency of large fragment vector is efficiently improved by electroporation .
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AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.
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Objective To construct Gadd45a expression plasmid and induce its expression in human T cells.Methods Gadd45a was amplified by reverse transcription PCR from human embryonic stem cells,and cloned into the pcDNA3.1 vector.The recombinant plasmid or blank plasmid was transfected into Jurkat cells or normal human CD4+T cells using electroporation,and the expression of Gadd45a was detected by quantitative RT-PCR and Western blot.Results Human Gadd45a expression plasmid was constructed successfully.Gadd45a was overexpressed both in Jurkat cells and normal human CD4+T cells after these cells were transfected with pcDNA3.1-Gadd45a.Conclusion The construction of Gadd45a expression plasmid and induction of Gadd45a overexpression in human T cells lay the foundation for further research on the role of Gadd45a in the epigenetic mechanism.
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Objective To construct multiple myeloma mucin MUC1-2VNTR gene eukaryotic expressing vector,which provided the basic material for further study of multiple myeloma DNA vaccine.Methods MUC1-2VNTR coding gene as target gene,and a KOZAK sequence was inserted before it.Hind Ⅲ and Xba Ⅰ restriction enzyme site were inserted on both ends.Then the whole sequence was synthesized and cloned into pcDNA3.1/myc-his B vector,and the recombinant vector was identified by restriction enzyme digestion and DNA sequencing.Results Synthesized MUC1-2VNTR gene was 140 bp.Restriction enzyme digestion and DNA sequencing confirmed pcDNA3.1/MUC1-2VNTR/myc-his B including the whole exact translation frame region and MUC1-2VNTR gene.Condnsion The pcDNA3.1/MUC1-2VNTR/myc-his B has been successfully constructed,which provides the basic material for further studies of MUC1 mucin function and multiple myloma DNA vaccine.
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In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 (+) to generate pcDNA3. 1 (+)/HA and pcDNA3.1 (+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1 (+)/HA and pcDNA3.1 (+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 (+)/HA or pcDNA3. 1 (+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.
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[Objective]To observe the expression of osteogenic growth peptide(OGP) gene,which had been transfected into rabbit bone marrow stromal cells(BMSC) by adenovirus,and detect the proliferation and differentiation of rabbit bone marrow stromal cells after gene transfection in vitro.[Methods]pcDNA3.1-OGP was constructed by using gene clone and recombined technique.With the help of lipofectamine 2000,BMSC were transfected with pcDNA3.1-OGP.The positive cell clones were selected with G418.The expression of OGP gene was observed by RT-PCR.Alkaline phosphatase(ALP) activity and type I collagen in pcDNA3.1-OGP transfected bone marrow stromal cells were measured to observe the differentiation from bone marrow stromal cells into osteoblast lineage.[Results]The pcDNA3.1-OGP vector was constructed successful.According to analysis of RT-PCR,alkaline phosphatase(ALP) and type I collagen assay showed that OGP gene could be expressed in BMSC,and could induce bone marrow stromal cells differentiation into osteoblast lineage.[Conclusion]The pcDNA3.1-OGP vector can be constructed successfully and its expression is confirmed in BMSC.The expression of OGP in bone marrow stromal cells can induce BMSC differentiation into osteoblast lineage.
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Objective To establish a cell line stably expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases. Methods Eukaryotic expression vector pcDNA3.1(+)TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay. Results Eukaryotic expression vector pcDNA3.1(+)TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the hours). Conclusion The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.
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Objective:To investigate the effect of pcDNA3.1 vector on house keeping gene expression and set guide to further study work in using pcDNA3.1. Methods:5 cell lines were transfected with pcDNA3.1,48 h post transfection total RNA was extracted and applied for real time PCR to detect the RNA level of GAPDH、 ?-actin、18sRNA difference.Meanwhile,extract total protein and analyze the GAPDH and?-actin expression by Western blot.Results:RNA and protein levels all show of a decrease in 5 cell lines after transfection,with the most obviously ?-actin.Conclusion:pcDNA3.1 might have non specifinhibition effect to cell,when using pcDNA3. 1 for gene function research,it is not recommend to use ?- actin as reference gene.
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Objective To explore the relation of c jun antisense gene transfection and cardiomyocyte apoptosis following hypoxia and burn serum treatment Methods Burn serum was collected from Wistar rats with 30% total body surface area(TBSA) Ⅲ degree burn Rats inhaling mixed gas containing 1% O 2 was used as hypoxia model The c jun antisense gene recombinant was constructed by genetic recombination technique Cardiomyocytes from neonatal Wistar rats were cultured in vitro with hypoxia and burn serum treatment c jun antisense gene recombinant was transfected into the cultured cardiomyocytes Cardiomyocytes were stained with TUNEL for the examination of cardiomyocyte apoptosis at 12, 24 and 48 h after hypoxia and burn serum treatment In addition, the number of apoptotic cardiomyocytes was counted The results were processed statistically Results In the group with only the addition of burn serum and hypoxia(non transfected group), the numbers of apoptotic cardiomyocytes(mean per high power visual field) were 7 1?0 842, 28 4?1 635 and 13 2?1 525, respectively But after transfection of c jun antisense gene recombinant, numbers of apoptotic cardiomyocytes were 4 1?0 716,12 3?1 455 and 8 5?1 341, respectively There was a significant difference between the transfected group and the non transfected group( P
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Objective To explore the effect of c fos antisense gene transfection on the protection of cardiomyocytes following hypoxia and burn serum treatment. Methods Burn serum was collected from Wistar rats with 30% total body surface area(TBSA) of Ⅲ degree burns. The mixture gas containing 1% O 2 was used as hypoxia model. The c fos antisense gene recombinant was constructed by genetic recombination technique. Cardiomyocytes from neonatal Wistar rats were cultured in vitro with hypoxia and burn serum treatment. c fos antisense gene recombinant was transfected into the cultured cardiomyocytes. Expression of c fos mRNA was determined by RT PCR. Expressions of c fos protein, troponin T and ? Tubulin in cardiomyocytes were determined by Western blotting in the transfected and non transfected groups. Results RT PCR results showed that the expression of c fos mRNA increased significantly in the non transfected group. But after transfection of c fos antisense gene recombinant, the expression of c fos mRNA decreased significantly as compared with the non transfected cardiomyocytes. Western blotting results showed that the expression of c fos protein in the transfected group decreased remarkably as compared with the non transfected group, but the expressions of ? Tubulin and troponin T increased significantly in the transfected group. Conclusion Burn serum and hypoxia can cause the injury of cardiomyocytes. c fos antisense gene recombinant transfection has the protective effect on cardiomyocytes exposed to burn serum and hypoxia.
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Objective To establish the expression of exogenous tissue plasminogen activator (TPA) gene in human umbilical vein endotheliocytes to provide both theoretical basis and new method for gene therapy of ischemic heart disease and prevention of postoperative vessel re-stenosis. Methods Expression vector pcDNA3 1(+)TPA was constructed, and transfected into human umbilical vein endotheliocytes cultivated in vitro by lyposome. The exogenous TPA expression was observed. Results The expression vector pcDNA3 1(+)TPA could efficaciously express TPA in human umbilical vein endotheliocytes. The protein quantity of TPA in the transfected cells was 568 6ng/10 6cell/24h, when measured by enzyme-linked immunosorbent assay, while that in the non-transfected cells was 17 8ng/10 6 cell/24h. The exogenous TPA's activity in the transfected cells was 108 8IU/10 6cell/24h, when measured by chromogenic substrate assay, while that in the non-transfected cells was 5 6 IU/10 6cell/24h. Conclusion When pcDNA3 1(+)TPA was transfected into human umbilical vein endotheliocytes, exogenous TPA could be expressed efficaciously, which provides theoretical basis and new method for TPA gene therapy of ischemic heart disease.
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Objective To investigate the immune efficacy of nucleic acid vaccination in rabbits against Schistosoma japonicum with pcDNA3 1(+)/MLP(SJP) Methods The 24 New Zealand rabbits were randomly divided into 2 groups. The rabbits of experimental group were vaccinated by each quadriceps muscle of leg with pcDNA3 1(+)/MLP nucleic acid vaccination and the control group rabbits were vaccinated with pcDNA3 1(+). Each rabbit was immunized four times with 2 weeks interval. The rabbits were challenged 2 weeks after final DNA boosting by percutaneous infection with cercariae. Sixty days after infection the rabbits were sacrificed, the livers were investigated, and the worms and eggs in livers were counted. Blood sera were collected from rabbits and investigated. Results In the experimental group,the egg reduction rate was 28 10%. The rabbits of experimental group produced IgA, IgG 1, INF-?. Conclusion DNA vaccination with pcDNA3.1(+)/MLP could induce partial protective immunity against Schistosoma japonicum in rabbits.
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Objective:To construct and identify the recombinant eukaryotic plasmid PcDNA3.1-MxA encoding MxA with the eukaryotic plasmid PcDNA3.1.Methods:Recombinant plasmid PAdTrack-MxA constructed before and PcDNA3.1 were amplified in Escherchia coli JM109.Plasmids were double-digested with restrictive endonucleases NotⅠ and XbaⅠ after extracted,and then ligated to construct recombinant eukaryotic plasmid PcDNA3.1-MxA.The recombinant plasmid was selected for Ampicillin resistance and then confirmed by digestion with NotⅠ and XbaⅠ,PCR and sequencing.The sequence was homology-analysed compared with the sequence of MxA gene in PAdTrack-MxA and the sequence of MxA gene published in Genebank(NM_002462) with sofeware DNAssist 2.0.Results:The restrictive endonuclease and PCR analysis confirmed that the recombinant eukaryotic plasmid PcDNA3.1-MxA was constructed successfully.Sequencing analysis revealed that the cloned segment was 2012bp including MxA gene sequence,and the nucleotide sequence of MxA gene was same to that in PAdTrack-MxA exactly.There were five variations in nucleotide sequence compared with published sequence in Genebank,which caused only one amino acid residue replaced from isoleucine to valine.This replaced amino acid residue was located in the non functional area of MxA protein.Conclusions:The recombinant eukaryotic plasmid PcDNA3.1-MxA is constructed successfully,which lays foundation for the further study on anti-HBV effects of MxA.
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Objective:To construct plasmid coexpressing GM-CSF and HBsAg,enhance hepatitis B DNA vaccines antivirus effect.Methods:(1)The HBsAg-encoding fragment and GM-CSF-encoding fragment were created by PCR amplification from pEcob6 and pCD-hGM-CSF respectively,then were cloned into plasmid pcDNA3.1(+),pcDNA3.1-S and pcDNA3.1-GM-CSF(without stop codon)were constructed.(2)HBsAg DNA fragment (with stop codon) was amplified by PCR,then was subcloned into plasmid pcDNA3.1-GM-CSF.Results:Recombinant pcDNA3.1-S and pcDNA3.1-GM-CSF-S were confirmed by using restriction enzymes and DNA sequencing.HBsAg was detected in the lysates and supernatants from cells transfected with pcDNA3.1-S and in the lysates from cells transfected with pcDNA3.1-GM-CSF-S.Conclusion:Two eukaryotic expression plasmids were constructed successfully and could express HBsAg.
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0.05).Conclusion:The pcDNA3.1/hTSHR has been successfully constructed.Animal models of Graves' disease have been made successfully by genetic immunization.