Inhibition of RAW264.7 Macrophage Inflammatory Cytokines Release by Small Haparin RNAi Targeting TLR4

In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-αreleased by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-αrelease by RAW264.7 cells evoked by LPS.     

Construction and Expression of Bivalent Membrane-anchored DNA Vaccine Encoding Sj14FABP and Sj26GST Genes

In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 (IL-2) upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human plaeental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj 14. The expression of Sl14 and Sl26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and pIRES-Sj26-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.     

Construction and expression of the bicistronic expression vector with RANTES and SDF-1 genes

Objective: To construct bicistronic expression vector with RANTES and SDF-1 genes, the ligands of HIV-1 principal coreceptors, and identify its expression. Methods: RANTES-KDEL was amplified from plasmid pCMV-R-Kby PCR and cloned into eukaryotic expression vector pCMV-S/K. Gene transfection into HeLa cells was carried out by lipofectin. Indirect immumofluorescence and radioimmunoprecipitation were used to confirm the expression of RANTES and SDF-1. Results:The construction of pCMV-R-K-S-K was confirmed by enzymatic digestion and sequencing.RANTES and SDF-1 were shown expressed in HeLa cells by indirect immumofluorescence and radioimmunoprecipitation. Conclusion: pCMV-R-K-S-K was constructed and expressed in cell line Hela successfully, which will contribute to further study of gene therapy of AIDS by HIV-1 coreceptors knockout.     

Construction of a Targeting Adenoviral Vector Carrying Survivin Promoter for Expressing EGFP Gene in Laryngeal Carcinoma Cell Line Hep-2

In gene therapy for any type of malignancy, tumor specificity is of utmost importance. Here we constructed the adenoviral vector bringing EGFP gene under the control of survivin promoter for evaluation of the possibility of target gene therapy in laryngeal carcinoma. First, Survivin promoter （SP） was amplified by PCR in the replace of CMV promoter in the plasmid pShuttle. Enhanced green fluorescent protein gene （EGFP） digested from the plasmid pEGFP-CI was cloned into plasmid pShuttle. Further two genes were cloned to Adeno-X Viral DNA. The recombinant adenoviral plasmid was transferred to HEK293 cells by lipofectamine. After titrating the virus, the laryngeal carcinoma cells （Hep-2） were transfected by the adenovirus and the expression of EGFP gene was detected. The recombinant Ad-SP-EGFP was correctly constructed and confirmed by restriction endonuclease analysis and DNA sequencing. After Hep-2 cells were transfected by the virus, RT-PCR showed that survivin mRNA was transcribed from the tumor cells. Western blot and fluorescent microscope showed EGFP protein was expressed in transgene Hep-2 cells. Present results suggest that survivin promoter may provide a new promising tool for target gene therapy of human malignancies.     

Construction and Expression of Bivalent Membrane-anchored DNA Vaccine Encoding Sj14FABP and Sj26GST Gene

In order to construct a eukaryotic co-expression plasmid containing membrane-anchored Sjc14FABP and Sjc26GST genes and identify their expression in vitro, Sj 14 and Sj26 genes were ob- tained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sjl4 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human interleukin-2 （IL-2） upstream Sj 14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyi-terminal of human placental alkaline phosphatase （PLAP） downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The two modified genes were altogether cloned into a eukaryotic co-expression plasmid plRES, resulting in another new recombinant plasmid plRES-Sj26-Sj14. The expression of Sj 14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays （IFA） when the plasmid plRES-Sj26-Sj 14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj 14 were successfully constructed and the expression of modified Sj 14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj 14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes.     

Construction and identification of recombinant lentivirus-mediated gene transfer system for rat transducer of regulated CREB activity 1

Objective:To construct a recombinant lentivirus vector which carries SD rat transducer of regulated CREB activity-1(TORC1) gene and examine its ability to express the TORC1 gene in vitro.Methods:The coding sequence of SD rat TORC1 gene was amplified using PCR and cloned into pGC-FU vector.293T cells were transfected using Lipofectamine 2000 and packaged for the recombinant lentivirus particles.When the cloned sequence was identified to be right,the recombinant lentivirus particles were amplified in a large quantity.The titer of virus was determined by real-time PCR and the level of TORC1 expression was examined by Western blot. Results:The recombinant lentivirus vector carrying TORC1 was constructed successfully and could express TORC1 at a high level in 293T cells in vitro,and the titer determined by real-time PCR was 2×108 TU/ml.Conclusion:The recombinant lentivirus vector could express TORC1 gene at a high level,and was very helpful in the study of exploring the effect of TORC1 on spinal cord injury.     

Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.     

Expression and Purification of SARS Coronavirus Membrane Protein

To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported. The recombinants were transformed into Escherichia coli (E. Coli) BL21 (DE3) and induced by Isopropyl-β-D-thiogalactopyranoside (IPTG). The expression of 27 kD (1 kD=0. 992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography. Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence. This provided the basis for the further study on SARS virus vaccine and diagnostic agents.     

Expression of the human era cDNA in E.coli

Objective: To amplify human era (Hera) gene, then express it in E.coli. Methods: Human era gene, after amplified by PCR and identified by sequencing, was inserted into the expression vector pGEX-4T3 in which exogenous gene was controlled by Ptac promoter. The recombinant plasmid pGEX-Hera was transformed into DH5 ( and induced with IPTG chemically. Results: The human era gene was amplified and the sequence was correct. When the bacteria with pGEX-Hera was induced, an anticipated 65 000 protein band appeared on SDS-PAGE gel and amounted to 23% of total bacterial protein. Conclusion: The human era gene has been successfully amplified and efficiently expressed in E.coli.     

A cell-based high-throughput screening assay for Farnesoid X receptor agonists

Objective To develop a high-throughput screening assay for Farnesoid X receptor （FXR） agonists based on mammalian one-hybrid system （a chimera receptor gene system） for the purpose of identifying new lead compounds for dyslipidaemia drug from the chemical library. Methods cDNA encoding the human FXR ligand binding domain （LBD） was amplified by RT-PCR from a human liver total mRNA and fused to the DNA binding domain （DBD） of yeast GAL4 of pBIND to construct a GAL4-FXR （LBD） chimera expression plasmid. Five copies of the GAL4 DNA binding site were synthesized and inserted into upstream of the SV40 promoter of pGL3-promoter vector to construct a reporter plasmid pG5-SV40 Luc. The assay was developed by transient co-transfection with pG5-SV40 Luc reporter plasmid and pBIND-FXR-LBD （189-472） chimera expression plasmid. Results After optimization, CDCA, a FXR natural agonist, could induce expression of the luciferase gene in a dose-dependent manner, and had a signal/noise ratio of 10 and Z＇ factor value of 0.65, Conclusion A stable and sensitive cell-based high-throughput screening model can be used in high-throughput screening for FXR agonists from the synthetic and natural compound library.     

Cloning of NHE-1 gene fragment from human lung can.cer cells and construction of its antisense expression vector

Objective: To clone the partial sequence of Na^ /H^ exchanger- 1 (NHE- 1) gene of human lung cancer cells and insert it reversely into the multiclone site of pLXSN in order to construct an antisense expression vector for tumor gene therapy it~ vivo. Methods: With use of the upstream and downstream primers containing Barn H I and EcoR I in their 5‘ ends respectively, a partial sequence of the first exon of NHE-1 gene was cloned in a length of 454 bp from genomic DNA of human lung cancer cell A549 with PCR method. The product was then direetionally and reversely insert into the multiclone site of pLXSN. Finally, the constructed recombinant was identified with agarose gel electrophoresis and DNA sequencing. Results: The cloned fragment was 461 bp in length and successfully ligated to pLXSN with the identification by agarose gel etectrophoresis. DNA sequencing confirmed that the fragment cloned and inserted into the vector was identical with the targeted one. Conclusion: The targeted fragment is successfully cloned and reversely inserted into pLXSN in our experiment. The antisense expression vector of NHE-1, pNHE-1, was constructed successfully.     

Experimental study on MyoD gene induced differentiation of bone marrow mesen-chymal stem cells into myoblasts in vitro

Objective: To explore the possibility of the transfection of MyoD gene induced bone marrow mesenchymal stem cells ( MSCs) to differentiate into myoblasts in vitro. Methods: The eukaryotic expression plasmid vector pIRES2-EGFP-MyoD was transfected into MSCs with lipotransfection method, and the positive cells were selected by G418; The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was identified by sequencing; The reporter gene enhanced green fluorescence protein ( EFGP) was observed in the transfected cells under a fluorescent and a laser confocal microscopes; Immunohistochemical methods was used to examine the expressions of MyoD, myogenin, myosin, myoglobin and desmin in the differentiated cells. The ultrastructure changes of the cells before and after transfection were observed with electron microscopy. Results: The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was as same in sequence a