The design of DNA-based alphavirus vectors significantly improves the utility of these replicon vectors. The DNA-based replicon vectors can be used in expressing foreign genes and preparing RVP in virto efficiently, also in developing replicon vaccines and gene therapy vectors in vivo. The approach involved the conversion a RNA-based replicon vector into a layered DNA-based replicon vector by the RNA polymerase Ⅱ promoter and transcription termination/polyadenylation signal transcribed replicon RNA from DNA. When DNA-based alphavirus vector tranfected into cells, the first layer includes a eukaryotic RNA polymerase Ⅱ expression cassette that initiates transcription of RNA in nucleus. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the RNA vector corresponds to virus RNA replication cycle and results in high level expression of foreign gene. DNA and RNA-based bifunctional replicon expression vector pSCTA and helper vector pSHCTA were successfully constructed by replacing the SP6 promoter used in the original system pSFV1 and pSFV-helper2 derived from Semliki Forest virus (SFV) with CMV promoter and T7 promoter, and inserting BGH transcription termination and polyadenylation signal downstream 3′-untranslated region (UTR). In order to obtain DNA-based highly efficient replicon vectors, they were further modified to construct additional three DNA-based SFV replicon expression vectors and corresponding helper vectors. To investigate the efficiency of foreign gene expression level by the four different DNA-based SFV expression vectors and recombinant virus particle (RVP) prepared by cotranfecting with corresponding helper vectors, improved DNA-based replicon vectors pSCAR and pSHCAR derived from SFV were developed. high level protein could be generated using the new vector system by transfecting DNA into BHK21 cells and High titer of RVP produced by cotranfecting with helper vector. Antigen genes were also expressed in cells by the replicon expression vector. Additionally, reporter gene expression was observed in mice muscle following injection with SFV DNA vector. Anti-?-Gal antibody response and cell-mediated immune response were induced after intramuscular inoculation of the ?-Gal-encoding SFV replicon DNA. The results suggested that highly efficient DNA-based replicon vectors pSCAR and pSHCAR were constructed by modifying the SFV vectors. The improved DNA-based replicon vectors enhance the utility of them, and can be developed as potentially replicon vaccines and gene therapy vectors.

Objective To explore the immunogenicity of recombinant chimeric 6Aβ15-T including the Aβ1-15 epitope and a T-helper epitope formulated with different adjuvants and to evaluate its feasibility as a candidate vaccine for Alzheimer disease (AD).Methods The recombinant chimeric antigen 6Aβ15-T formulated with Al adjuvant, Freund′s adjuvant or MF59 adjuvant was administered to two strains of mice .The 6Aβ15-T-immunized group without adjuvants ( Mock) and non-immunized group (Control) were included in this study as control groups .The specific antibody and cellular immune response of the chimeric antigen were evaluated .Results In BALB/c strain mice, three types of adjuvants could substan-tially boost the immunogenicity of chimeric antigen 6Aβ15-T and produce a high level of specific-Aβ(β-amyloid) antibod-ies.In C57BL/6 strain mice, the existence of adjuvants enhanced the immune response of 6Aβ15-T antigen, but the mice in Mock group also produced a strong antibody response .In two strains of mice, prevalence of anti-AβIgG1, which was an indicator of Th2 polarization, was observed in the 6Aβ15-T-immunized mice.Additionally, the Al adjuvant induced a high-er level of IgG1 antibody titers, and the ratio of IgG1/IgG2a was the largest.As expected, the 6Aβ15-T antigen formulated with or without adjuvants induced PADRE-specific, but not Aβ42-specific T cellular immune response .Conclusion The 6Aβ15-T antigens formulated with different types of adjuvants could induce strong Th 2-polarized Aβ42-specific antibody re-sponses without activating self-reactive Aβ42-specific T cells in two strains of mice .The results suggested that the recombi-nant chimeric antigen 6Aβ15-T is a good candidate vaccine for AD .

Objietive Observe the clinical curative effect of PPH in treatment of complexity hemorrhoids and circular hemorrhoid. Methods From January 2010 to January 2014, the clinical data of 278 patients who underwent PPH to treat complexity hemorrhoids, mixed hemorrhoid and circular hemorrhoid were retrospectively analyzed. The postoperative recent and forward curative effect was observed. Results The average operativetime was 28 minutes, and the average hospital stay were 6. 1 days. There were postoperative complications such as pain, anal skin edema, bleeding and urinary retention. 186 cases were cured (66. 9%),35 cases were markedly improved (12. 6%),20 ca-ses were of poor effect (7. 2%),and recurrence occured in 37 cases (13. 3%). Conclusion PPH has the advantages of simple in operation and quick in postoperative recovery. But there were to many postoperative complications and the recurrence rate is high. So PPH needs more further observation and study.

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.
Artificial Gene Fusion ; CD4 Antigens ; biosynthesis ; genetics ; Chemokine CCL20 ; biosynthesis ; genetics ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; physiology ; Humans ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, HIV ; antagonists & inhibitors ; Transfection ; fms-Like Tyrosine Kinase 3 ; biosynthesis ; genetics

To investigate the cell-killing effect and its possible mechanism of rClone30-hDR5 in combination with TRAIL on human hepatic carcinoma (HCC) cell line, first of all, recombinant plasmid pee12.4-hDR5 was introduced into HepG2 cells by liposome transfection. After five rounds of screening by flow cytometry, HepG2 cells expressing high levels of DR5 on cell surface were isolated. The cytotoxicity of TRAIL to selected cells was higher than that of TRAIL to HepG2 cells by MTT method (P < 0.01). The result suggested that the cloned hDR5 gene had biological activity. MTT assay showed that, rClone30- hDR5 in combination with TRAIL more efficiently inhibited the tumor growth of HepG2 cells compared to rClone30-hDR5 or TRAIL in vitro. The results of Annexin V-FITC/PI staining and Quantitative Real-time PCR indicated that rClone30-hDR5 in combination with TRAIL significantly increased the mRNA levels of caspase 3 and caspase 8, and induced the apoptosis of tumor cells. HepG2 cells were infected with rClone30-hDR5 or rClone30 at MOI of 1. The expression of hDR5 on tumor surface increased significantly by rClone30-hDR5 compared to that by rClone30, which contributed to the sensitivity to TRAIL. In conclusion, rClone30-hDR5 in combination with TRAIL has potential application value in cancer treatment.

In order to enhance the antitumor efficacy of recombinant Newcastle disease virus, rNDV-IL15 was rescued in this study. Recombinant plasmid prNDV-IL15 was constructed, and BHK21 cells were transfected with the recombinant plasmid. Finally, the recombinant Newcastle disease virus rNDV-IL15 was successfully rescued. The growth curves of these two recombinant viruses were determined. Murine melanoma B16F10 cells were infected with rNDV-IL15 at MOI of 0.1, and the expression level of IL15 in the supernatant was detected by ELISA. The antitumor efficacy of rNDV-IL15 and rNDV was compared in vitro and in vivo. Results showed that prNDV-IL15 was constructed and recombinant virus rNDV-IL15 was successfully rescued. The growth curve of rNDV-IL15 showed that the growth of rNDV-IL15 had not been changed after insertion of IL15 gene. Results showed that there was high level of IL15 expression in the supernatant of rNDV-IL5-infected B16F10 cells (1 044.3 +/- 27.7 ng x mL(-1)). rNDV-IL15 and rNDV significantly inhibited the growth of B16F10 cells in vitro in a time-dependent manner. However, there was no significant difference between them. In animal experiments, rNDV-IL15 efficiently suppressed tumor growth in vivo when compared with rNDV, and the difference was statistically significant. The results suggested that rNDV-IL15 is a more effective antitumor agent.

Objective To evaluate the influence of data quality on the sensitivity of early warning syndromic surveillance system based on medical institutions in Qianjiang,Hubei province and explore the relationship between data quality and sensitivity of early warning of the system.Methods The delay reporting rate and underreporting rate were calculated for the evaluation of the data quality.Data obtained from semi-synthetic simulated outbreak and area under the curve (AUC) were used in combination to test the sensitivity of early warning of various models and select the optimal model.Time-series generalized additive model (GAM) was used to analyze the curve fitting and threshold effect between data quality and early warning sensitivity of the system.Results A total of 179 905 cases were reported from April 1,2012 to January 31,2014,in which 8 744 were not reported timely (16.45％).Averagely 416 reporting were delayed in each month.There were 2 566 cases which were underreported (4.83％).Compared with other early warning models,i.e.Cumulative Sum (CUSUM),Shewhart,Exponentially Weighted Moving Average (EWMA),Early Aberration Reporting System (EARS-3C),the MA model had the maximum area under the curve (AUC=0.93),and the difference was significant (P＜0.001).The early warning sensitivity ranged from 84.89％ to 97.25％ during the operation period of the syndromic surveillance system.Underreporting had influence on early warning sensitivity,when underreporting rate was over 2.78％,the sensitivity would decrease obviously.No obvious associations were observed between the delay reporting rate and early warning sensitivity of the system.Conclusion The data quality had influence on the early warning sensitivity of the syndromic surveillance system based on medical institution in Qianjiang.In the context of this study,underreporting had the main influence on the sensitivity of early warning.