The effects of TwHMGR overexpression on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii / 药学学报

Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase (TwHMGR) is an important regulation site in terpenoids metabolic pathway in cytoplasm which is the first speed limit enzyme of MVA pathway. In order to investigate the effects of TwHMGR on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii, the overexpression of TwHMGR (OE-HMGR) was studied in this paper. We cloned the full-length of TwHMGR to construct overexpression vector by Gateway technology then delivered the expression vector into Tripterygium wilfordii suspension cells by gene gun. qRT-PCR was used to detect the expression of TwHMGR:the expression of TwHMGR was increased to 1.75 folds over the control group (empty vector:pH7WG2D) in the overexpression group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that:the contents of triptolide and celastrol were increased to 163.93% and 190.04% of over the control group in the overexpression group. Based on these findings, the positive effect on the accumulation of active terpenoids, triptolide and celastrol in Tripterygium wilfordii was found and the results laid a foundation of the synthetic biology research on important active terpenoids in Tripterygium wilfordii.

ICOSL could upregulate immune response in Graves′s disease animal models / 中国免疫学杂志

Objective:To study the pathological mechanism of the inducible co-stimulator molecular and ligand ( ICOS/ICOSL) in Graves disease animal.Methods:45 out-bred BALB/c mice were randomly divided into three groups with 15 rats in each group;using gene gun to deliver different plasmid injection.Group A was delivered with pCDNA3.0-mICOSL and pCDNA3.0-hTSHR, Group B with pCDNA3.0-hTSHR and null pCDNA3.0 with Group C for immunization as the control group.The concentration of serum free thyroxine immunization was deter mined with immunoassay and serum thyrotropin receptor antibody ( TRAb ) with ELISA, supernatant of IFN-γconcentration in mouse spleen cells was measured with radioimmunoassay,and hTSHR transected CHO cells were incubated to detect the concentration of cAMP to deter mine autoantibody TRAb activity.Results: After plasmid injection serum FT4 level in Group A (0.49±0.25) pg/ml ( q=6.571,P=0.023) was higher than that in Group C,the standard rate was higher than Group B and C (χ2=14.47,P=0.005).IFN-γconcentration of mice spleen cultured supernatant in Group A (1.88±0.41) pmol/L was significantly higher than the other two groups.The activity of autoantibody TRAb in Group A 188.3 (179.7-260.2) %was higher than that in the other two groups ( P=0.027 ) .Conclusion: Exogenous delivery of pCDNA3.0-mICOSL plasmid in GD mice could stimulate the spleen lymphocytes to secrete more IFN-γ,increase the activity of TRAb autoantibodies and might lead to upregulation of immune response in Graves animal model in vivo.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70 percent of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50 percent of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.

CD8+ T-cell Activation in Mice Injected with a Plasmid DNA Vaccine Encoding AMA-1 of the Reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.

Construction and primary application of Sh-2K~d-HBc tetramer / 中国免疫学杂志

Objective:To prepare and test tetrameric sH-2K~d-HBc complex for the further measurement of the specific CTL response.Methods:PE labled streptavidin with 4 biotinylated binding sites can bind to 4 biotinylated monomer to form the corresponding tetramer.Mice were immunized via different methods of genetic immunization by use of the construted pcDNA3-C plasmid to get the specific CTLs.Then our prepared tetramer was applied to stain the specific CTLs by the analysis of flow cytometry.Results:We applied our prepared tetramer to stain the cells from the experimental groups and control group.The results showed the tetramer was able to discriminate the frequencies of specific CTL induced by the three immunol methods(0.24%,0.26%,0.36% vs 0.07%,P≤0.05).This demonstrated that the prepared tetramer could bind its targets specifically and efficiently.The three immunol methods induced different levels of immune responses.Compared with the traditional muscle injection,gene gun induced weaker humoral immune response and stronger cellular immune response,and hydrodynamic injection induced the strongest humoral and cellular immune responses.Conclusion:Have successfully constructed the sH-2K~d-HBc tetramer.The techniques and methods can be used for preparation of tetramers of other types of MHCⅠ molecules.

Effect of gene gun transduction of K-RAS Antisense gene on the expression of K-RAS P21 protein in human pancreatic carcinoma cells / 基础医学与临床

Objective To observe the effect of gene gun transduction of K-RAS Antisense gene on the expression of K-RAS P21 protein in human pancreatic carcinoma cells.Methods K-RAS Antisense gene was transduced into pancreatic carcinoma cells by gene gun transduction, the expression of K-RAS P21 protein on BxPC-3、AsPC-1 and MiaPaCa-2 pancreatic carcinoma cells line were examined by western blot and immunocytochemistry staining. Results The expression of K-RAS P21 protein in AsPC-1 and MiaPaCa-2 pancreatic carcinoma cells was obviously lower after the transduction of specific K-RAS Antisense gene,which has little impact on BxPC-3 pancreatic carcinoma cells. Conclusion Gene gun transduction of K-RAS Antisense gene is a potential method for treatment of pancreatic carcinoma.

In Vivo Transfection of Rabbit Synovium using the Particle Bombardment Method / 대한정형외과연구학회지

Treatment of arthritis with the gene-transfering technique is one of tile important applications of gene therapy in orthopedics. The synovium can be transfected with the genes producing therapeutic proteins. Among the numerous methods of transfering genes, particle bombardment or gene gun is one of the most versatile tools and has the advantage of easy accessibility and efficiency. The authors planed to transfer the reporter gene into the synovium in vivo and confirm the expression of the gene. Cold particle microcarriers to deliver the DNA were coated with plasmids containing human placental alkali no phosphatase cDNA as a marker gene. The microcarriers were projected with the aid of the hand-held gene gun system into the rabbit knee synovium. After three days, the synovium was harvested and frozen sections of the tissue were made. The expression of the reporter gene in the synovium was confirmed with alkaline phoshatase staining. In summary, the authors have shown that the reporter gene was successfully delivered into the rabbit knee synovium in vivo with particle bombardment method and transient expression of the gene was confirmed with special staining. Our study suggest that in vivo transfection of the synovium using tile gene gun can be applied as a novel method for treating arthritis.

The Establishment of Gene Gun-Mediated Human GM-CSF Gene Transfection System and It′s Stable Expression in Tumor Cells / 中国肿瘤生物治疗杂志

Objective: To provide an effective hGM CSF gene transferring vector mediated by gene gun and a basis for study of hGM CSF gene modified tumor cell vaccines. Method: The gastric tumor cell line (SGC) was transfected with eukarytic expression plasmid deoxyribonucleic acid containing the human granulocyte macrophage colony stimulating (hGM CSF) gene using the gene gun. The SGC cell clones (SGC GM CSF 1～5)secreting high hGM CSF level were obtained after G418 resistance selection. The hGM CSF gene had been integrated into chromatosome of SGC by the assay of RT PCR.Results: There was hGM CSF production whose lane was about 30 kD in the culture medium of SGC GM CSF by the assay of SDS PAGE and Western blot. SGC GM CSF had the ability of the high level of GM CSF for a long time(mean 247ng/(10 6 cell?24 h).Conclusion: The hGM CSF gene transferring vector mediated by gene gun was effective and safe. These results provide a basis for study of GM CSF gene therapy for cancer.

Construction of DNA Vaccine of Trichinella spiralis Muscle Larvae and Its Expression in Mice / 中国寄生虫学与寄生虫病杂志

Objective To construct and express recombinant plasmid containing the structural gene encoding {Mr 31 000} antigen of Trichinella spiralis muscle larvae. MethodsThe target gene TspE1 was amplified by RT_PCR, cloned into pUC18 vector, and sub_cloned into the eukaryotic expression vector pcDNA3. BALB/c mice were immunized with the purified recombinant plasmid pcDNA3_TspE1 by gene_gun bombardment. The expression of recombinant plasmid in the skin tissue was observed by HE staining and immunohistochemical staining. Results and Conclusion The recombinant plasmid pcDNA3_TspE1 was successfully constructed and expressed in the BALB/c mice.