Construction of eukaryotic expression plasmid of human promyelocytic leukemia gene of six transcripts and subcellular location analysis of its recombinant proteins / 中国生物制品学杂志

@#Objective To construct eukaryotic expression plasmids of human promyelocytic leukaemia(hPML) gene of six transcripts and analyze the subcellular location of the recombinant proteins.Methods Primers were designed according to the hPML gene sequences registered in GenBank databases.Six transcripts of hPML gene fragments(hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were amplified by RT-PCR,which were linked to the eukaryotic expression vector pCAGGS respectively.The obtained eukaryotic expression plasmids of six transcripts of hPML gene were transfected into 293T cells respectively and detected for their protein expression by Western blot,while transfected into Vero cells and detected for their subcellular location by indirect immunofluorescence assay(IFA).Results The target gene fragments of the six eukaryotic expression plasmids were consistent with the hPML gene sequences registered in GenBank.All the six recombinant proteins showed specific binding with Myc antibody,among which the recombinant protein hPML Ⅰ,Ⅱ,Ⅳ,Ⅴ and Ⅵ were located in the nucleus and cytoplasm,while the recombinant protein hPML Ⅶ was mainly located in the cytoplasm,rarely in the nucleus.Conclusion The eukaryotic expression plasmids of six transcripts of hPML gene all can be expressed correctly in mammalian cells,and the expressed recombinant proteins were located in nucleus and cytoplasm simultaneously or mainly in cytoplasm.This study provides an experimental basis for subsequent study on the antiviral and other biological functions of recombinant protein hPML.

Therapeutic effects of IL-6 and BAFF fusion protein eukaryotic expression plasmid on animal model of Sj?gren′s syndrome / 中华微生物学和免疫学杂志

Objective:To investigate the effects of a eukaryotic expression plasmid for IL-6 and B-cell activating factor (BAFF) fusion protein on the histopathological changes in salivary and lacrimal glands of non-obese diabetic (NOD) mice with Sj?gren′s syndrome and to elucidate the possible therapeutic mechanism of IL-6/BAFF fusion protein eukaryotic expression plasmid in NOD mice.Methods:The eukaryotic expression plasmid for IL-6/BAFF fusion protein was constructed. After transfecting CHO cells with the plasmid, the expression of IL-6/BAFF fusion protein was detected by Western blot. BALB/c mice were injected with the plasmid every two weeks for three times and the titers of anti-IL-6 and anti-BAFF antibodies were measured by ELISA. Twenty-one NOD mice were randomly divided into three groups (control group, empty vector group and therapy group) by numerical table method. The mice in the therapy group were injected with the IL-6/BAFF fusion protein eukaryotic expression plasmid once a week for six times and the mice in the empty vector group were injected with empty plasmid. The levels of anti-IL-6 and anti-BAFF antibodies as well as cytokines (IL-6, BAFF, INF-γ, IL-10 and IL-17A) in mouse serum samples were detected by ELISA. The proportions of Th17, Treg, Th1 and Th2 cells in mouse splenocytes were measured by flow cytometry. Focal lymphocyte infiltration and pathological changes in the lacrimal and salivary glands of mice were observed under light microscopy after HE staining.Results:The eukaryotic expression plasmid for IL-6/BAFF fusion protein increased the levels of anti-IL-6 and anti-BAFF antibodies in the serum of BALB/c mice ( P<0.05). The levels of anti-IL-6 and anti-BAFF antibodies in the serum of NOD mice in the therapy group increased ( P<0.01), while the expression of IL-6, BAFF, INF-γ, IL-10 and IL-17A in NOD mice in the therapy group was lower than that in the control group and the empty vector group ( P<0.05). The percentages of Treg and Th2 cells in the splenocytes of NOD mice increased after treatment ( P<0.05). Moreover, the eukaryotic expression plasmid for IL-6/BAFF fusion protein significantly improved the irregular size and morphology of glandular vesicles in the lacrimal and salivary glands, reduced the ductal dilatation and decreased the focal lymphocyte infiltration in NOD mice. Conclusions:The eukaryotic expression plasmid for IL-6/BAFF fusion protein induced the production of anti-IL-6 and anti-BAFF antibodies, decreased the expression of inflammatory cytokines, regulated the balance of Th17/Treg and Th1/Th2 cells, improved the irregular alveolar structure and ductal dilation in the lacrimal and salivary glands and reduced the focal lymphocyte infiltration in NOD mice. This study showed that eukaryotic expression plasmid for IL-6/BAFF fusion protein might serve as a potential target for therapeutic targeting of T and B cells.

Construction of recombinant eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes / 国际眼科杂志(Guoji Yanke Zazhi)

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.

Effect of VEGF-C Gene Transfection on Expression of VEGF-C in Human Breast Cancer MCF-7 Cell / 中国普外基础与临床杂志

Objective To explore the effect of vascular endothelial growth factor-C (VEGF-C) gene transfection on the expression level of VEGF-C in human breast cancer MCF-7 cell. Methods The constructed VEGF-C gene eukaryotic expression vector was transfected into the human breast cancer MCF-7 cell by using lipofectamine transfection reagents, and the positive cell clones were obtained through G418 selection after transfection. The expressions of VEGF-C mRNA and protein were detected by RT-PCR and Western blot respectively. Results Following the transfection of the VEGF-C recombination plasmid, there were significant differences on the expression levels of VEGF-C mRNA and protein between pcDNA3.1-VEGF-C transfection group and pcDNA3.1 transfection group (12.382?2.183 vs 6.039?1.950, P

Construction of pEGFP-N1-Fcy::Fur recombinant plasmid and observation of its expression in ovarian cancer cells / 重庆医科大学学报

Objective:To construct a EGFP(Enhanced green fluorescent protein)-labled euk-aryotic expression plasmid of Fcy::Fur suicide gene and to detect its expression in SKOV3 cell line. Methods:With the technology of gene re-arrangement,Fcy::Fur gene in pORF-Fcy:Fur plasmid was subcloned into pEGFP-N1 vector,with its correctness evaluated by the means of r-estriction enzyme analysis and sequencing.It was transfected into SKOV3 cells with lipofectin,the transient expression of GFP was observed under flu- orescence microscope after 24 hours and detected by Western blot. Results:Correct construction of pEGFP-N1-Fcy::Fur was identi- fied by methods of restriction enzyme analysis and nucleotide sequence determination.A total of 60% transfe-cted cells emitted out green fluorescence under fluorescent microscope after 24 h after transfecti-.on. Fcy::Fur gene expressed by the transfected cells were testified by Western blot. Conclusion:The recombinant eukaryotic expression vectors have been constructed successfully and effec- tive-ly expressed in ovarian cancer cells,which may provide an experimental basis for gene therapy of ovarian cancer.

Bioinformatics analysis and construction of eukaryotic expression plasmid of Cx50 V64G mutation / 国际眼科杂志(Guoji Yanke Zazhi)

AIM : To construct and analyze eukaryotic expression plasmid inserted by Cx50 with V64G mutation through bioinformatics software.METHODS: The full coding domain sequence of Cx50 with V64G mutation was acquired from the blood of patients with cataract and was cloned into pcDNA3.1 /Amp (+).The constructed plasmid was identified with PCR , enzyme digestion and sequencing. The analysis of Cx50 with V64G mutation was performed with bioinformatics software.RESULTS : Cx50 with V64G mutation was successfully amplified and its eukaryotic expression plasmid was constructed. Valine-64 is well conserved in the first extracellular loop of connexin 50 in different species and also in different human α -type gap junctional proteins.CONCLUSION : The successive reconstruction and verification of eukaryotic expression plasmid containing Cx50 with V64G mutation established the foundation for further studying the mechanism of cataract.

Construction of a Novel Eukaryotic Expression Plasmid pcDNA6/myc-his-EGFP Band Its Applications in Expression of Recombinant Genes / 中国生物工程杂志

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

Construction of a Novel Eukaryotic Expression Plasmid pcDNA6/myc-his-EGFP Band Its Applications in Expression of Recombinant Genes / 中国生物工程杂志

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

Construction of a Novel Eukaryotic Expression Plasmid pcDNA6/myc-his-EGFP Band Its Applications in Expression of Recombinant Genes / 中国生物工程杂志

Enhanced green fluorescent protein( EGFP), myc epitope and polyhistidine metal-binding tag are often used as a marker for recombinant fusion protein in many gene expression vectors, each marker has its own function, EGFP emits green fluorescence for direct detection, myc epitope facilitates recombinant fusion protein detection using its antibodies, polyhistidine tag allows purification of recombinant fusion protein using resin.Hitherto, no a plasmid vector can integrate all of these functions. In this study we constructed a novel eukaryotic expressive plasmid, designated as pcDNA6/myc-his-EGFP B, which integrated the functions of EGFP, myc epitope and polyhistidine tag. Importantly, a linker octo - peptide in N terminal of EGFP was designed using LINKER program. A DNA fragment encoding a putative protein containing a signal peptide of human interleukin 2(IL-2) in N terminal was cloned into pcDNA6/myc-his-EGFP B in frame with the C-terminal peptide to construct pMHES. 2.2.15 Cells were transfected with pcDNA6/myc-his-EGFP B and pMHES, and Balb/c mice were intravenously injected with pcDNA6/myc-his-EGFP B by tails, results revealed that both of the plasmids worked in 2.2.15 Cells and livers of Balb/c mice. Assuming gene of the IL-2 was inserted into pcDNA6/myc-his -EGFP B in frame with EGFP, myc and 6 × His, three-dimensional structure for this putative expression product was simulated using Modeller8V2, results revealed that IL-2, EGFP, myc and 6 × his did not interfere each other and octo- peptide linker owned certain flexibility. The results suggest that pcDNA6/myc-his-EGFP B may be useful as a genetic tool for mammalian cells and a vector for gene therapy.

Construction of eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG and its expression in vitro / 第三军医大学学报

Objective To construct eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG,and to detect the expression of the plasmid in vitro.Methods hOPG cDNA fragment was extracted from plasmid MGC:29565,amplified by PCR,and inserted into pcDNA3.1(-)vector,then identified by restriction endonuclease digestion and nucleotide sequencing.C2C12 cells were transfected with the plasmid using Lipofectamine 2000.The expression of OPG protein was detected by immunohistochemistry techniques and bone wafer pit assay.Results The constructed recombinant plasmid contained the sequence of hOPG gene.After transfection with the plasmid,active OPG protein could be expressed in C2C12 cells.Conclusion The constructed eukaryotic expression bicistron plasmid vector pcDNA3.1-hOPG can express OPG in vitro,which is the basis for further study on the treatment of bone absorption caused by abnormal activity of osteoclasts.

Construction of eukaryotic expression plasmid encoding Fba protein of group A streptococcus and elicitation of immune responses by it / 中国免疫学杂志

Objective:To construct eukaryotic expression plasmid ecording a novel surface protein Fba of GAS, and to explore its impact on host immune responses.Methods:Fba gene was amplified by PCR using strain SSI-9 (GAS M1 serotype isolates) as the template, then cloned into pcDNA3.1 for constructing eukaryotic expression plasmid pcDNA3.1/fba and sequenced. Female CD1 mice were randomly individed into 6 groups, and immunized respectively with Fba protein, M protein, pcDNA3.1/fba + Fba protein, pcDNA3.1/fba, pcDNA3.1 and PBS as control. Blood was obtained from the mice and specific antibody of IgG was detected by ELISA. Spleen cells were assessed with lymphocyte proliferation assays.CD4+T cell and CD8+T cell were detected by flow cytometry (FCM). Assay results were analyzed with SPSS10.0.Results:The IgG against Fba protein kept hightest levels in group immunized with Fba protein. The levels of lymphocyte proliferation, CD4+, CD8+T cell were significantly high in the group pcDNA3.1/fba. Conclusion:(1) Just as M protein, the antibody to Fba protein could be efficiently induced by immunization with Fba protein, which showed that Fba protein was hopefully to be a candidate protein for vaccine against GAS.(2) Eukaryotic expression plasmid pcDNA3.1/fba was successfully constructed and this recombinant plasmid could efficiently induce antibody and CD4+ T cell for againsting GAS.

The biological effect of Fas Ligand transfected HepG2 cell / 中国免疫学杂志

Objective:To evaluate the expression of apoptosis related gene Fas Ligand(FasL) in human hepatocellular carcinoma(HCC) cells HepG2 and its significance in apoptosis.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1hisB- FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells were detected by immune histochemistry. After stained by annexin V and propidium iodine, cells were passed throw flow cytometer and examined by fluorescence microscope and sym-focus laser scaning microscope.Results:In comparison with untransfected cells,the soluble FasL could be detected in the supernatant of transfected cells, Fas L can be expressed in the membrane and cytoplasm of transfected cells. The apoptotic cell rate in transfected cells was 36.30%, as the control, untransfected cells was 11.53%.Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine stain.Conclusion:This supports a novel pathway of HCC cells were apoptotic itself via the CD95-CD95 ligand system without involvement of immune cells.

CONSTRUCTION OF HYBRID NUCLEIC ACID VACCINES BASED ON PLASMODIUM FALCIPARUM MEROZOITE SURFACE PROTEIN1BLOCK17REGION / 中国寄生虫学与寄生虫病杂志

AIM:To construct hybrid nucleic acid vaccines which contain Plasmodium falciparum merozoite surface protein 1 (MSP1 ) block 1 7gene and gene fragment coding for several T cell epitopes. METHODS:MSP1 block 1 7gene of FUP strain was connected with a polyvalent gene fragment which contains several T cell epitopes from MSA1 ,MSA2 ,RESA,IL- 1 and Tetanus toxin,the connected gene fragment (hybrid gene,HG) was cloned into eukaryotic expression plasmid p CDNA3.1 (- ) ,VR1 0 1 2 for intracellular expression,VR1 0 1 2 /TPA for secretion,the recombinants were identified by PCR and restriction enzyme digestion. RE- SUL TS &CONCLUSION:The hybrid nucieic acid vaccines:VR1 0 1 2 /HG,VR1 0 1 2 /TPA/ HG and p CDNA3.1 /HG were successfully constructed.