Construction and characterization of a eukaryotic expression vector of full-length and mature IL-37b / 中国比较医学杂志

Objective To construct a eukaryotic expression vector pEGFP N1/IL-37b of full-length and mature IL-37b,and to detect the expression of both full-length and mature IL-37b in RAW 264.7 cells, a mouse macrophage cell line. Methods To construct the eukaryotic vectors of full-length and mature IL-37b by using plasmid pUBC/IL-37b as a template containing the coding region of IL-37b full-length gene. To detect the expression of IL-37b by western blot and confocal microscopy after transfected the recombinant plasmid into RAW 264.7 cells, and to detect the inhibition of full-length and mature IL-37b on IL-6 production by real-time PCR. Results Eukaryotic vectors pEGFP N1/IL-37b expressed full-length and mature IL-37b after transfection in cells, which inhibited LPS-induced IL-6 production. Conclusions Eukaryotic vectors of full-length and mature IL-37b can be successfully constructed,and lays a foundation for further study of anti-inflammation functions and mechanisms of IL-37b.

Role of B7-H6 over-expression in NK cell-mediated apoptosis of hepato-cytes / 中国病理生理杂志

AIM:To investigate the role of B7 homologue 6 (B7-H6) over-expression in natural killer (NK) cell-mediated hepatocyte apoptosis. METHODS:The full-length fragment of B7-H6 gene was amplified by PCR and sub-cloned into linearized eukaryotic expression vector pIRES2-EGFP to construct recombinant B7-H6 over-expression vector pIRES2-EGFP-B7-H6. The recombinant plasmid was identified by double digestion, PCR and sequencing, and was then transfected into L02 cells. The expression of EGFP was observed by fluorescence microscopy and the transfection efficiency was evaluated by flow cytometry. B7-H6 expression was confirmed by qRT-PCR and Western blot. The L02 cells transfect-ed with pIRES2-EGFP-B7-H6 recombinant plasmid were co-cultured with NK-92 cells at different effector/target ratios,and the cytotoxicity of NK-92 cells was evaluated by CCK-8 assay.RESULTS:The strong green fluorescence in the L02 cells was observed under fluorescence microscope 48 h after transfection. The transfection efficiency reached 92.6%. The ex-pression of B7-H6 at mRNA and protein levels was remarkably increased 48 h after transfection. The cytotoxicity of NK-92 cells against L02 cells transfected with pIRES2-EGFP-B7-H6 plasmid was significantly higher than that of the null vector transfection group (P<0.05).CONCLUSION:The recombinant eukaryotic expression vector pIRES2-EGFP-B7-H6 was constructed successfully. The cytotoxic effect of NK-92 cells against L02 cells can be enhanced by transfecting L02 cells with pIRES2-EGFP-B7-H6 plasmid.

Construction of rat beta defensin-2 eukaryotic expression vector and expression in the transfected rat corneal epithelial cell / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To construct a recombinant eukaryotic expression vector of rat beta defensin-2(rBD-2), transfect it into the rat corneal epithelial cells with lipofection, determine the expression of target gene in the transfected cells, and discuss the potentiality of recombinant plasmid expressed in corneal epithelial cells, hoping to provide an experimental foundation for further study on the antimicrobial activity of rBD-2 <i>in vitro </i>and <i>in vivo</i> and to assess the probability of defensins as a new application for infectious corneal diseases in the future. <p>METHODS: The synthetic rBD-2 DNA fragment was inserted between the <i>Xho</i>I and <i>BamHI</i> restriction enzyme cutting sites of eukaryotic expression vector pIRES2-ZsGreen1 to construct the recombinant plasmid pIRES2-ZsGreen1-rBD-2, then transformed it into <i>E.coli DH5α</i>, positive clones were screened by kanamycin and identified with restriction endonucleases and sequencing analysis. Transfection into the rat corneal epithelial cells was performed by lipofection. Then the experiment was divided into three groups: rat corneal epithelial cell was transfected with the recombinant plasmid pIRES2- ZsGreen1-rBD-2, rat corneal epithelial cell was transfected with the empty plasmid pIRES2-ZsGreen1 and the non-transfected group. The inverted fluorescence microscope was used to observe the transfection process. At last, the level of rBD-2 mRNA expressed in the transfected cells and the control groups are compared by the real-time fluoresence relative quantitative PCR. <p>RESULTS: The recombinant eukaryotic expression vector of pIRES2-ZsGreen1-rBD-2 was successfully constructed. The level of rBD-2 mRNA in transfected cells was significantly higher than that in control groups through the real-time fluorescence relative quantitative PCR. <p>CONCLUSION: The recombinant eukaryotic expression vector pIRES2-ZsGreen1-rBD-2 could be transfected into rat corneal epithelial cells, and exogenous rBD-2 gene could be transcripted into mRNA in the transfected cells.

Construction and expression of eukaryotic expression vector of human IL-37 b gene / 中国实验动物学报

Objective To construct the eukaryotic expression vector pEGFP-N1/IL-37b and analyze the expression of IL-37 gene in THP-1 cells. Methods Total RNA was extracted from human peripheral blood mononuclear cells ( PB-MCs) and the coding region of IL-37b gene was amplified by RT-qPCR. Then, the gene was cloned into pEGFP-N1 eu-karyotic expression vector. After transfected the recombinant plasmid into THP-1 cells, the expression of IL-37 was detec-ted by RT-qPCR and Western blot. Results Double restriction enzyme digestion and gene sequencing showed that IL-37b gene was correctly inserted into the eukaryotic expression vector pEGFP-N1. RT-qPCR and Western blot showed that the IL-37 expression level was increased significantly (P<0. 01) after transfection in THP-1 cells. Conclusions We successful-ly constructed a novel anti-inflammatory cytokine IL-37 eukaryotic expression vector pEGFP-N1/IL-37b, which lays a foun-dation for further study on IL-37 functions and its association with related diseases.

Eukaryotic expression of human NOVA1 protein and identification of its anti-hypoxia activity / 生物工程学报

The aim of this study was to construct the eukaryotic expression vector of pCMV-Myc-NOVA1 based on NOVA1 gene, and to screen the optimum expression condition after transfecting to PC12 cells, and further to explore the distribution of NOVA1 protein in PC12 cells using cell immunohistochemistry, and to identifyits anti-hypoxia activity. According to the NOVA1 gene sequence of NCBI database, we designed the upstream and downstream primers, and performed polymerase chain reaction (PCR) to amplify the full length cDNA coding sequence using pCR4-TOPO-NOVA1 as a template. The products were digested by restriction endonuclease SalⅠand XhoⅠ, and conjugated to the eukaryotic expression vector ofpCMV-Myc followed by validating by digestion and direct sequencing. Subsequently, the validated pCMV-Myc-NOVA1 was transfected to PC12 cells followed by optimizing of transfection ratio and transfection time, and identified by qPCR, Western blotting and cell immunohistochemistry respectively. After validation by digestion and direct sequencing, the eukaryotic expression vector of pCMV-Myc-NOVA1 was correctly constructed. The optimum transfection ratio of plasmid to Lipo 2000 was 1:2.5, and the optimum transfection time was 72 h. At the optimum transfection condition, the expression level of NOVA1 mRNA and protein significantly increased, and after transfection of pCMV-Myc-NOVA1, NOVA1 protein mainly distributed in cell nucleus and cytoplasm. After 6 h hypoxia, the cell proliferation activity was significantly increased compared to that of the control and pCMV-Myc group. Our findings provided a reference for exploring the mechanism of NOVA1, and also a technical support for potential drug development of NOVA1.

shRNAs targeting high mobility group box-1 lead to inhibition of E-selectin expression via homeobox A9 in human umbilical vein endothelial cells / 中华危重病急救医学

ObjectiveTo approach the regulatory mechanism of high mobility group box-1 (HMGB1) on the expression of E-selectin in human umbilical vein endothelial cell (HUVEC).Methods Homeobox A9 (HOXA9) siRNA was transfected to HUVEC at logarithmic phase, real-time fluorescence quantitative polymerase chain reaction (real-time qPCR) and Western Blot were used to determine the HOXA9 mRNA expression and protein expressions; a blank control group and a nonsilence negative control group were set. HUVEC stable transfected with pRNA-u6.1/Neo-HMGB1 shRNA plasmids (HUVEC with low-expression HMGB1) was obtained, and HOXA9 and E-selectin mRNA expressions were determined with real-time qPCR; a nonsilence transfection group served as the negative control. The HOXA9 siRNA was transfected to HUVEC with low-expression HMGB1 as co-transfection group, and the E-selectin expressions was determined with real-time qPCR; a HMGB1 shRNA group and a HOXA9 nonsilence group served as control.Results① HOXA9 mRNA (2-ΔΔCT) and protein expression (integralA value) in blank control group were 1.094±0.115 and 1.031±0.060. Compared with nonsilence transfection group, HOXA9 siRNA transfection group could significantly reduced mRNA and protein expression of HOXA9 [HOXA9 mRNA (2-ΔΔCT): 0.257±0.030 vs. 1.035±0.091,t = 14.010,P = 0.002; HOXA9 protein (integralA value): 0.278±0.042 vs. 0.975±0.014,t = 27.310, P = 0.002].② Compared with nonsilence transfection group, HMGB1 shRNA transfection could up-regulate HOXA9 mRNA expression in HUVEC (2-ΔΔCT: 2.519±0.278 vs. 0.856±0.063,t = 10.100,P = 0.001), also could down-regulate E-selectin mRNA expression (0.311±0.046 vs. 1.080±0.201,t = 7.415,P = 0.000).③ Compared with HOXA9 nonsilence group and HMGB1 shRNA group, HMGB1 shRNA and HOXA9 siRNA co-transfected HUVEC cells could significantly elevate E-selectin mRNA expression (2-ΔΔCT: 3.445±0.428 vs. 1.085±0.212, 1.004±0.104,t1 = 8.507, t2 = 9.603, bothP< 0.001).Conclusion HMGB1 may regulate E-selectin expression through the HOXA9 regulation in HUVEC.

Construction of human BTG2 eukaryotic expression vector with FLAG tag and its expression in HeLa cells / 吉林大学学报(医学版)

Objective To construct an eukaryotic expression vector of human B-cell translocation gene 2 (BTG2), to express the FLAG-tagged BTG2 protein in HeLa cells,and to supply an experimental tool for investigating the function of BTG2 gene.Methods The full-length BTG2 fragment was obtained by PCR and inserted into the multiple cloning site of pcDNA3.1 (+)vector. Oligo DNA encoding FLAG tag was designed and inserted into pcDNA3.1(+)-BTG2 to construct another vector pcDNA3.1(+)-FLAG-BTG2.The HeLa cells were divided into pcDNA3.1(+)empty vector group,pcDNA3.1(+)-BTG2 group and pcDNA3.1(+)-FLAG-BTG2 group.The HeLa cells were transfected with recombinant plasmids.Western blotting using anti-FLAG antibody was performed to detect the expression of FLAG-BTG2 protein in HeLa cells.Results The sequence of the vector was verified by both BamH Ⅰ endonuclese digestion and DNA sequencing. The Western blotting analysis confirmed that FLAG-fused BTG2 was detected in pcDNA3.1(+)-FLAG-BTG2 group but not in empty vector or pcDNA3.1(+)-BTG2 groups. Conclusion The eukaryotic expression vector pcDNA3.1(+)-FLAG-BTG2 is successfully constructed and FLAG-tagged BTG2 protein is expressed in HeLa cells.

Construction of eukaryotic expression vector of mouse microRNA miR-21 and identification its expression activity in 293 cells / 局解手术学杂志

Objective To construct the eukaryotic expression vector for mouse microRNA miR-21 and identification its expression activ-ity in 293 cells. Methods The genomic sequence containing pre-miR-21 was amplified from mouse genomic DNA by PCR and cloned into the pRC/CMV plasmid. The constructed recombinant plasmid pRC/CMV-mmu-miR-21 was transfected to 293 cells by lipofectamine 2000, and the stably transfected cells were screened with G418,from which total RNA was extracted for detecting the expression of mature miR-21 by northern blot. In the meantime,a luciferase report plasmid examing the activity of miR-21 named pmiR-21-Luc reporter was also construc-ted,and luciferase activity analysis indicated the product of pRC/CMV-mmu-miR-21 indeed had biological activity. Results Both restriction enzyme digestion analysis and sequencing proved the recombinant plasmids were constructed correctly. The miR-21 was highly expressed in the screened clones of 293 cells and it had good biological activity. Conclusion The eukaryotic expression plasmid of mouse miR-21 was successfully constructed,which laid the foundation of further investigation of the role of miR-21 during skin wound healing.

Construction and expression of HSV1-tk eukaryotic vector in lung adenocarcinoma AGZY cell line / 实用肿瘤学杂志

Objective The purpose of this study is to construct eukaryotic gene vector of herpes simplex virus type 1 thymidine kinase(HSV1-tk)and to observe the expression of HSV1-tk in lung adenocarcinoma AGZY cell line.Methods The full length HSV1-tk gene was amplified by PCR from plasmid pHSV 106 and was inserted into pMD18-T.The recombinant plasmid was recombined with eukaryotic vector plRES 2-EGFP u-sing gene recombinant technique .HSV1 -tk was transfected into adenocarcinoma AGZY cell line with Lipo-fectamineTM 2 000.Fluorescence microscopy was used to detect the transfection and expression of HSV 1-tk.RT-PCR was used to detect the mRNA levels of HSV 1-tk.The cell proliferation was measured by MTT assay .Re-sults A length of 1 130 bp gene sequence was obtained by PCR .The expressions of HSV 1-tk at mRNA and protein levels were displayed by RT -PCR and Western blot .MTT analysis showed that there were no significant changes cell survival on after transfection .Conclusion The eukaryotic expression vector of HSV 1 -tk report gene is successfully constructed and HSV 1-tk is effectively expressed in transfected AGZY cells .

Construction of Hi FGF2 eukaryotic expression plasmids and its over-expression induced cell apoptosis / 中国药理学通报

Aim To construct eukaryotic expressing plasmid of hi FGF2 ( high molecular weight isoform fi-broblast growth factor-2,hi FGF2) gene and to investi-gate its effect on apoptosis after its overexpression in HEK293 cells. Methods The DNA template primer was designed and synthesized. The pDsRed1-N1 plas-mids were digested by the restriction enzymes of Nhel and Hind III. The hi FGF2 was ligated with linearized pDsRed1-N1 by T4 DNA Ligase. The recombinant plasmid was identified by endonuclease digestion and sequenced. The recombinant hi FGF2 plasmid was transient transfected into HEK293 cells by Lipofectami-neTM 2000 Reagent. The transfection efficiency was de-tected by fluorescence inversion microscope. The cell apoptosis was detected by Annexin V-FITC/PI apopto-sis detection kit with flow cytometry analysis. Results The pDsRed1-N1 eukaryotic expression vector was consistent with the design. The recombinant hi FGF2 plasmid was transfected in HEK293 cells. The trans-fection rate was more than 70%. The FITC/PI dyeing rate in hi-FGF2 over-expression HEK297 cells was a-bout ( 29. 12 ± 2. 81 )%. Conclusions pDsRed1-N1 eukaryotic expression vector is successfully constructed and transfected into HEK293 cells. Over-expression of hi FGF2 induces cell apoptosis.

Melanoma antigen A3 sourced from laryngeal cancer was expressed in mouse melanoma cell model / 中国免疫学杂志

Objective:To construct tumor cell model by determination of the pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid expressing steadily in mouse melanoma B16 cells.Methods:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid being constructed from the melanoma-associated antigen A3 genes sourcing laryngocarcinoma in advance was translated into the mouse melanoma B16 cells under the mediation of lipofectamine,and the positive clones were detected with G418.The expression of enhanced green fluorescent protein( EGFP) and MAGE-A3 mRNA in positive clones were detected by fluorescence microscopy and fluorescence quantitative PCR ( qRT-PCR ) assay, respectively.Results:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected into B16 cells successfully, the green fluorescence of fusion protein expression was found, and MAGE-A3 mRNA transcription in B16 cells expressions were detected in positive clones.Conclusion:The pIRES2-EGFP/MAGE-A3 eukaryotic expression plasmid has been transfected effectively and expressed stably by liposome method in the B16 cells.The expression of MAGE-A3 tumor cell model has been successfully established,which provide data for the study of laryngocarcinoma immunotherapy.

Construction of Tre enzyme eukaryotic expression vector and identification of its function in loxLTR sequence-specific recognition / 医学研究生学报

Objective Clearing HIV provirus is the key to cure AIDS .The study was to construct the Tre enzyme eukaryotic expression vector and identify its function in specific recognition of loxLTR sequence in HIV provirus . Methods Tre gene was in-serted into eukaryotic expression vector pcDNA 3.1 gene recombination manipulation by genetic recombination techniques including gene synthesis , PCR, restriction enzyme digestion and ligation .EGFPpA-LoxLTR sequence was inserted into pmCherry-N1 vector and was tested by restriction enzyme digestion , PCR and sequencing .Constructed vectors were electroporated into HeLa cells , then using fluorescence microscopy to observe fluorescence intensity changes . Results PCR, restriction enzyme digestion , electrophoresis and sequencing confirmed that Tre enzyme eukaryotic expression vector had been constructed successfully , and it could specifically recog-nize and cut loxLTR sequence after being transfected into Hela cells . Conclusion Constructed Tre enzyme eukaryotic expression vector can be expressed in Hela cells and specifically recognize loxLTR sequence , which has prepared the experimental ground for fur-ther studies of clearing HIV provirus .

Transfection and expression of leptin gene in human placental mesenchymal stem cells / 实用口腔医学杂志

Objective:To construct a eukaryotic expression vector of leptin gene,and to transfect it into human placental mesenchy-mal stem cells(HPMSCs)and appraise its expression.Methods:Primers were designed and the leptin gene was obtained by RT-PCR from human adipose tissue.The aimed segments were inserted into the eukaryotic expression vector pIRES2-EGFP,plasmid pIRES2-EGFP-LEP was constructed and identified by restricted enzymatic resection,and then transfected it into HPMSCs by liposome.The ex-pression of leptin in the transfected cells was detected by RT-PCR and Western blotting,the multi-differentiation potential of the cells was indentified.Results:The length of specific fragment was 500 bp,the recombinant plasmid pIRES2-EGFP-LEP presented 5.3 kb and 500 bp bands by restriction enzyme digestion.Leptin gene was expressed in transfected HPMSCs and the transfected HPMSCs maintained multi-directional differentiation potential.Conclusion:The eukaryotic expression vector of leptin can be transfected and ex-pressed in HPMSCs.

Building and identification of prion gene eukaryotic expression vector of human / 重庆医学

Objective To constructan eukaryotic expression recombinant plasmid named pEGFP-N2-PRNP .Methods Total RNA was extracted from alzheimer (AD) disease peripheral blood ,and the PRNP gene was amplified by reverse transcription-poly-merase chain reaction(RT-PCR) .By using gene recombination technique ,human PRNP cDNA was inserted into retroviral vector pEGFP-N2 .The recombinant plasmid was identified by a pair of specified primers containing the restriction sites of Xho Ⅰ and EcoRⅠ .Results The PRNP gene could be obtained by RT-PCR ,the recombinant plasmid was identified by restriction endonucle-ase analysis ,PCR and sequence analysis ,and the expression vector pEGFP-N2-PRNP ,which could be stably expressed in SH-SY5Y cells .Conclusion The recombinant plasmid pEGFP-N2-PRNP is constructed successfully ,Which offers a basic for the further re-search on PRNP biological fuction .

Construction of LAT1 eukaryotic expression vector of C57 mouse and its effect on the Neuro-2a cell / 中国比较医学杂志

Objective To construct the lamino acid transporter 1 eukaryotic expression vector of C 57 mouse and to express the gene inNeuro-2atumor cells,and explore the effect of LAT1on proliferation and apoptosis of Neuro-2a cell. Methods The full-length LAT1 cDNA was synthesized by RT-PCR and cloned into pcDNA3.1vector to construct recombinant plasmid.The constructed pcDNA3.1-LAT1vector was verified by Enzyme digestion and sequencing and then transfected intomurine Neuro-2acellsby liposome.The transfected cells were selected with G418 and stably expressed strain was constructed .The expression of LAT1 was detected by RT-PCR and western blot .Proliferation was analyzed by MTT , cell cycle and apoptosis were detected by flow cytometric analysis .Results The full-length LAT1 cDNA was amplified successfully and pcDNA3.1-LAT1eukaryoticvector was constructed successfully .Enzyme digestion and sequencing confirmed the sequence was correct .Neuro-2acells were transfected and Stably expressed strain was constructed successfully.MTT showed that the group of transfected restructuring plasmid could significantly affect Neuro -2a cell proliferation more than the control groups ( P <0.05 ) .From the flowcytometric analysis , LAT1 could promote cell proliferation and inhibit Neuro-2a cell apoptosis.Conclusion LAT1 can express successfully inNeuro-2acells which were transfected with recombinantpcDNA3.1-LAT1plasmid.LAT1 in Neuro-2a cells can promote cell proliferation and inhibit the cell apoptosis which provides a basis for the study of LAT 1.That lays the foundation for studying biological effects of LAT 1.

Construction and verification of SIRT1 and its mutant T200I, E420K eukaryotic expression vector / 国际生物医学工程杂志

Objective To clone silent information regulator 1 (SIRT1) gene full-length cDNA,construct recombinant eukaryotic expression vector containing SIRT1 gene and its mutant T200I,E420K,so as to lay the foundation for further research of SIRT1 gene function.Methods RT-PCR amplified SIRT1 gene full-length cDNA.PCR products were cloned into the eukaryotic expression vector pcDNA3.1 (+) through double digestion and pcDNA3.1(+)-SIRT1 recombinant plasmid was obtained.Meanwhile,site-directed mutagenesis was applied to build its mutant pcDNA3.1 (+)-T200I and pcDNA3.1 (+)-E420K expression vector.Recombinant plasmid was identified by enzyme digestion and DNA sequencing and the recombinant eukaryotic expression vector of success was screened out.Results SIRT1 gene full-length cDNA was successfully cloned,and pcDNA3.1 (+)-SIRT1 eukaryotic expression vector and its mutant were also successfully constructed.Positive recombinant plasmid sequencing was compared after enzyme digestion,and it was completely consistent with the expected sequence.Transfected 293T cell line was established and HIS tagged SIRT1 protein was expressed.Conclusions We successfully constructed pcDNA3.1 (+)-SIRT1 and its mutant pcDNA3.1 (+)-T200I,pcDNA3.1 (+)-E420K eukaryotic expression vector,which may provide genetic material for biological function study of SIRT1 gene and its mutant T200I,E420K.

Construction and expression of eukaryotic expression vector of PEA-15 / 肿瘤研究与临床

Objective To construct the eukaryotic expression vector of pcDNA3.1-PEA-15 and express it in the human esophageal cancer (EC-109) cells,and to explore the effect of PEA-15 on EC-109 cells.Methods The PEA-15 gene was amplified from EC-109 by reverse transcriptase polymerase chain reaction (RT-PCR) and ligated to eukaryotic expression vector pcDNA3.1.After confirmation of recombinant plasmid was correctly by endonuc]eases digestion and DNA sequencing,the construct was transfected it into EC-109 through liposome inducing.The expression of PEA-15 in transfected EC-109 was detected by RT-PCR and Western blot.The cell growth inhibition ratio was evaluated by MTT assay.Results RT-PCR indicated that PEA-15 was highly expressed in EC-109 cells.The amplified fragment by RT-PCR was coincident with hypothesis enzyme digestion analysis and DNA sequencing confirmed that the pcDNA3.1-PEA-15 was constructed successfully.The expression of PEA-15 gene was increased obviously in the transfected EC-109 detected by RT-PCR and Western blot respectively (t =4.078,5.269,P ＜ 0.05).The cell growth inhibition ratio in the group which transfected pcDNA3.1-PEA-15 was significantly lower compared with the pcDNA3.1 transfect group after 72 hours (t =6.163,P ＜ 0.05).Conclusions The recombinant eukaryotic expression vector pcDNA3.1-PEA-15 is constructed successfully,and it can be expressed in EC-109.It also shows that PEA-15 has the function on cell growth which suggests that PEA-15 can inhibit the apoptosis of EC-109 cells and its expression involved in esophageal cancer development.

Construction of eukaryotic expression vector of UCA1 a(CUDR) gene and its expression in bladder cancer UM-UC-2 cells / 吉林大学学报(医学版)

Objective To construct an eukaryotic expression vector pcDNA-UCA1a(CUDR)and to observe its expression in bladder cancer UM-UC-2 cells, and to provide experimental basis for study on the relationship between UCA1a(CUDR)gene and bladder cancer.Methods Human total length of UCA1a(CUDR)gene was obtained from the 5′-RACE-Ready cDNA of bladder cancer BLZ-2 1 1 cells by PCR and was inserted into pcDNA3.1 (+)vector.pcDNA-UCA1a(CUDR)was identified by digestion with EcoRⅠ and BamHⅠ.The bladder cancer UM-UC-2 cells were transfected stably with the constructed eukaryotic expression vector pcDNA-UCA1a(CUDR). The expressions of UCA1a(CUDR)gene in the UM-UC-2 cells transfected with pcDNA-UCA1a(CUDR)and the UM-UC-2 cells transfected with pcDNA3.1(+)(control vector)were detected by RT-PCR.Results The inserted fragment with 2 200 bp was successfully amplified, which was in accordance with the expected results. The eukaryotic expression vector pcDNA-UCA1a(CUDR)was constructed successfully after identified by double enzyme digestion and sequencing.The RT-PCR results showed that the expression of UCA1a(CUDR)gene in the cells transfected with pcDNA-UCA1 a (CUDR ) was significantly increased compared with the cells transfected with pcDNA3.1 (+). Conclusion The eukaryotic expression vector pcDNA-UCA1a (CUDR ) is successfully constructed.The UCA1a(CUDR)gene highly expresses in the UM-UC-2 cells transfected with the expression vector.

Construction of eukaryotic expression vectors of FGFR3 gene and their expressions in human leukemia K562 cell line / 吉林大学学报(医学版)

Objective To construct the eukaryotic expression vectors of fibroblast growth factor receptor 3(FGFR3) MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN, and to detect their expressions in human chronic myeloid leukemia(CML)K562 cell line.Methods The full-length FGFR3 (fgfr3-WT)and dominant negative FGFR3 (fgfr3-DN)were amplified by polymerase chain reaction (PCR). The two genes were respectively digested with EcoRⅠand BamHⅠ,and then ligated into MSCV/puro to construct the recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN which were tranduced into K562 cells by LipofectaminTM 2000 after PCR,double digestion and DNA sequencing.The expressions of FGFR3 protein in K562 cells were detected by Western blotting and flow cytometry. Results The recombinant plasmids MSCV/puro-fgfr3-WT and MSCV/puro-fgfr3-DN were amplified by PCR method, and the results showed fgfr3-WT of 2 400 bp and fgfr3-DN of 1 200 bp had been successfully cloned into MSCV-puro vector. The 2 400 bp fragment was oblained after double digestion of recombinant plasmid.The sequencing results showed that the size of fgfr3-WT was 2 400 bp which was the same as the sequence from GeneBank.Fgfr3-DN of 1 200 bp was also in conformity with the expected sequence.Compared with control (K562 MSCV)group,the expression level of FGFR3-WT in MSCV/puro-fgfr3-WT transfection (K562-WT)group was increased to above 10 times.There was high expression of FGFR3-DN in MSCV/puro-FGFR3-DN transfection (K562-DN)group,but there was no expressions in control(K562 MSCV)group and K562-WT group.The flow cytometry results showed that the high expressions of FGFR3-WT were in 57.5% cells in K562-DN group and the high expressions of FGFR3-DN were in 41.5% cells in K562-DN group. Conclusion The K562 cell lines highly expressing FGFR3-WT and FGFR3-DN are constructed successfully.

Construction of Eukaryotic Expression Vector with HIV-1 Integrase for Expression in Hela Cells / 昆明医科大学学报

Objective To construct recombinant eukaryotic expression vector for EGFP fused HIV-1 integrase expression. Methods Wild type of HIV-1 integrase gene was cloned into eukaryotic expression vector-pcDNA6/V5-HisA. After restricted enzyme mapping, PCR confirmation and sequence confirmation, the recombinant plasmid was transfected into HeLa cells with Lipofectamine2000. After 24 hours, the expression of integrase was examined by immunofluorescence with confocal fluorescent microscopy. The cells were fixed with 4%paraformaledhyde. The cell nuclei were stained with Propidium Iodide (PI). Then the expression was imaged and analyzed with Confocal Microscopy. Results The integrase expressed significantly in HeLa cells in 24 hours after transfection. Integrase was expressed and localized into nuclei mainly. After fixed with 4% paraformaldehyde, the cell nuclei were stained with PI. When nuclei were showed in red in normal cells, the nuclei with integrase over expression turned yellow or orange. Conclusion The construction of eukaryotic expression vector of integrase was successful. Integrase was expressed and localized into nuclei mainly after transfection in HeLa cells.