Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

AIM: To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically. METHODS: Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP. The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP. PCR was used to detect the target gene fragments, and the viral titer was determined. A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR. CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL. CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay. RESULTS: The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully. Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus. PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome. The virus in the supernatant reached a titer of 1×10¹³ PFU/L. The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group. Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days. CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

AIM: To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma.METHODS: SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316-SSTR2.pDC316-SSTR2 was cotransfected with rescue plasmid pBHGlox (delta) E1, 3Cre into 293 cells by liposome reagent.Ad-SSTR2 was generated by site-specific recombination and confirmed by PCR.Ad-SSTR2 was propagated in 293 cells and purified.The titer of viral stock was determined by end-point dilution assay.Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan-2 was infected with recombinant adenovirus.RESULTS: pDC316-SSTR2 was successfully constructed.Recombinant adenovirus Ad-SSTR2 was acquired by pDC316-SSTR2 and pBHGlox (delta) E1, 3Cre cotransfected into 293 cells.Ad-SSTR2 was characterized by PCR.The virus titer was 6.0×1012 pfu/L.SSTR2 protein was detected after adenovirus infected capan-2 48 h with Western blotting.CONCLUSION: The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells.This investigation provides the basis for study of gene therapy of pancreatic carcinoma.     

Bone marrow mesenchymal stem cells modified by adenoviral vector containing CTLA4Ig inhibit immune response ex vivo

AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibited the lymphocyte proliferation ex vivo.     

Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

AIM:To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 (hFGF-10) gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS:HFGF-10 gene was amplified by PCR and ligated with shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hFGF-10, which was linearized with PmeI and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy-hFGF-10. The recombinant adenoviral plasmid was then transfected into HEK-293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF-10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirus on the proliferation of kerotinocytes was checked by MTT. RESULTS:The recombinant adenovirus containing hFGF-10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat cells reacted with hFGF-10 antibody. The recombinant adenovirus stimulated the proliferation of kerotinocytes. CONCLUSION:HaCat cells infected with the recombinant adenovirus expresses and secrets hFGF-10 protein, which promotes the proliferation of HaCat cells.     

Construction of recombinant adenovirus expressing BDNF and its expression in expanded rat mesenchymal stem cells in vitro

AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.     

Construction of recombinant adenoviruses expressing the helicobacter pylori CagA antigen

AIM: To construct a replication-defective recombinant adenovirus, which expresses the CagA gene. METHODS AND RESULTS: The CagA gene was amplified by PCR. This heterogeneous gene was cloned into shuttle vector pAdTrack-CMV. The recombinant adenovirus DNA was obtained by the homologous recombination between the shuttle vector and adenovirus DNA in E.coli 5183. After linearization， the recombinant adenovirus DNA was transfected into 293 cells and the recombinant adenovirus was obtained. Through this technique, the replication- defective recombinant adenovirus AdEasyCagA was constructed. CONCLUSIONS: The replication-defective recombinant adenoviruses AdEasyCagA was constructed successfully. The work will make a good foundation for studying the effects of the replication-defective recombinant adenovirus on Th1/Th2 balance in asthma and be useful for finding a new pathway to prevent and cure asthma.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.     

CTLA4Ig induces immune tolerance of T cells to oxidized-low density lipoprotein in vitro

AIM: Recently,it is widely accepted that atherosclerosis (AS) is an auto-immune related disease and the oxidized-low density lipoprotein (ox-LDL) is the most important AS-related antigen.In order to prevent immune injuries in AS and find new strategies to prevent AS,the immune tolerance of T cells to ox-LDL in vitro was induced in this study.METHODS: Human monocytes were separated from peripheral blood to induce dendritic cells (DCs).DCs were treated with LPS (30 μg/L),ox-LDL (10 mg/L) and LDL (10 mg/L) for 48 h.Then DCs were mixed with allogenic T lymphocytes to carry out mixed lymphocytes reaction (MLR).CTLA4Ig in different concentrations was added in the MLR of ox-LDL group.MTT method was used to assay the proliferation of T cells and expressed in stimulation index (IS).The CD25 expression and apoptosis of T cells in MLR were tested by flow cytometry.The excretion of IL-2,IFN-γ and IL-4 was assayed by ELISpot method.RESULTS: SI in ox-LDL group was higher than that in LDL group significantly (P<0.05) and CTLA4Ig inhibited the SI in ox-LDL group with dose-dependent effect (P<0.05,P<0.01).CTLA4Ig decreased the CD25 expression (P<0.05,P<0.01) and induced apoptosis of T cells in MLR (P<0.05,P<0.01).CTLA4Ig decreased the ELISpot counts of IL-2 and IFN-γ (P<0.01),while increased that of IL-4 (P<0.05).CONCLUSION: CTLA4Ig induces T cells tolerance to ox-LDL in vitro.CTLA4Ig inhibits T cells activation,promotes T cells apoptosis and Th1/Th2 immune deviation,which is the important mechanism in it′s induction of tolerance.     

Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.     

Cloning expression and toxic effect on the host bacterial viability of an outer envelope protein from the strong virulent leptospira lai in China

AIM: Construction of an eukaryote-E. coli shuttle expressing recombinant plasmid which expresses OmpL1 envelope protein of pathogenic Leptospira, serovar Lai strain 017. METHODS: The OmpL 1 gene was amplified by PCR from the leptospiral genome. Then it was cut with restriction enzymes and ligated to the plasmid pBK-CMV. The correct recombinant plasmid was screened out with analysis of restriction enzymes and PCR. After inducing the E. coli baring recombinant plasmid with IPTG,the complete protein of the bacteria was extracted for SDS-PAGE. At the same time, OD600 of the host bacteria was examined at different time after inducing or uninducing with IPTG. RESULTS: Five strains E. coli containing proper recombinant plasmids were screened out. Four strains E. coli expressed a new protein with a weight of 37 kD among them. With the expression of the heterogenous protein,the OD600 of the host bacteria decreased. CONCLUSION: The shuttle expressing plasmid of the OmpL 1 gene of strong virulent Leptospira strain 017 was successfully constructed. Furthermore,the recombinant plasmid expressed the expected OmpL 1 fusion protein in E. coli and the expression of the heterogenous protein had toxic effect on the host bacteria. This work was important for the future research of OmpL1 protein which relates to the diagnosis,new vaccine preparing and the pathogenic mechanism of leptospirosis.     

Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.     

Construction of siArid2 recombinant adenovirus and its effect on proliferation of hepatocarcinoma cells

AIM: To observe the effects of Arid2gene on cell proliferation and cell cycle by interference of endogenous Arid2 expression in hepatoma cells. METHODS: Three pairs of shRNA targeting Arid2gene were cloned into a shuttle vector to construct recombinant adenovirus plasmids. HEK293 cells were transfected with the recombinant adenovirus plasmids. After several rounds of the package and amplification, the high-titer adenoviruses AdsiArid2-1~3 were obtained. To verify the inhibitory effects of AdsiArid2 adenoviruses, Western blotting was used to detect the endogenous Arid2 protien expression in SMMC-7721 cells. Cell growth and cell cycle analysis were carried out by MTS assay and flow cytometry. RESULTS: High- titer recombinant adenovirus of siArid2 were successfully obtained, and named AdsiArid2-1~3, among which the AdsiArid2-3 had the best inhibitory effects. MTS assay showed that the absorbance values at 490 nm were increased at 72 h and 96 h after transduction compared with the mock and Adsicontrol groups. These data indicated that knockdown of Arid2 promoted the proliferation rate of SMMC-7721 cells(P<0.05). Moreover, the flow cytometry analysis revealed that the G1-phase distribution at 72 h in AdsiArid2 group was lower than that in mock group and Adsicontrol group. In contrast, the S-phase distribution in AdsiArid2 group was much higher than that in mock group and Adsicontrol group. CONCLUSION: The recombinant plasmids and recombinant adenovirus were successfully constructed. shRNA-mediated knockdown of Arid2 promotes the proliferation and the transition from G1 phase to S phase of hepatoma cells.     

Construction of eukaryotic expression vector and expression of outer membrane lipoprotein LipL41 gene of Leptospiria lai

AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.     

Role of B7-H6 over-expression in NK cell-mediated apoptosis of hepatocytes

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 subcloned 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 transfected 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 expression 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 mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.