Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease.</p><p><b>METHODS</b>The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis.</p><p><b>RESULTS</b>We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system.</p><p><b>CONCLUSION</b>The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.</p>

Effect of simian vacuolating virus 40 on development and differentiation of dendritic cells from Rhesus macaque / 病毒学报

To study the effect of simian vacuolating virus 40 (SV40) on development and differentiation of dendritic cells (DC) from rhesus macaque, the peripheral blood-derived dendritic cells from rhesus monkey were pulsed with inactivated SV40 and infective SV40, respectively at the 5th day post DC cultivation. Expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface at the 7th, 9th day post DC cultivation were analyzed by flow cytometry (FCM). The results showed that expressions of CD1a, HLA-DR, CD86 and CD83 on the cell surface in the inactivated SV40-pulsed experimental group were higher than those in the infective SV40-pulsed experimental group (P < 0.05). These cell surface molecules represented characteristic development and differentiation phase of DC. Down-regulation of expressions of these cell surface molecules indicated that infective SV40 might hamper differentiation and maturation of dendritic cells from rhesus monkey.

RNA interference-mediated inhibition of survivin expression in Hela cell line by siRNA expression vector targeting survivin gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare small interfering RNA (siRNA) targeting survivin for inhibition of endogenous survivin gene expression in Hela cell line and evaluate its effect on promoting Hela cell apoptosis.</p><p><b>METHODS</b>The recombinant plasmid pshRNA-survivin-1 and pshRNA-survivin-2 were constructed and transfected into Hela cells, in which the expression level of survivin was determined by immunofluorescence staining and survivin gene transcription detected by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>Introduction of the plasmids pshRNA-survivin-1 and pshRNA-survivin-2 into Hela cells resulted in efficient and specific inhibition of survivin expression as demonstrated by immunofluorescence staining. Semi-quantitative RT-PCR showed that mRNA transcription of survivin gene was reduced. In contrast, the control plasmid did not exhibit any inhibitory effect on the protein expression and mRNA transcription of survivin gene. PI-Annexin V staining indicated an apoptosis rate of the transfected Hela cells of (36.02-/+2.12)% (P<0.01) and (35.29-/+2.02)% (P<0.01), respectively.</p><p><b>CONCLUSION</b>The prepared siRNA targeting survivin gene is capable of inducing marked inhibitions of survivin protein expression and RNA transcription and significant enhancement of apoptosis in Hela cells, which shed light on a new strategy in gene silence therapy targeting survivin.</p>

Production in Pichia pastoris and characterization of genetic engineered chimeric HBV/HEV virus-like particles / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).</p><p><b>METHODS</b>The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.</p><p><b>RESULTS</b>The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.</p><p><b>CONCLUSION</b>The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.</p>

Construction of a recombinant human adenovirus expressing the ORF2 antigen of HEV and immunization of mice by mucosal system / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation.</p><p><b>METHODS</b>The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10.</p><p><b>RESULTS</b>Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response.</p><p><b>CONCLUSIONS</b>The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.</p>

Refolding and purification of the huGM-CSF(9-127)-IL-6(29-184) fusion protein / 生物工程学报

The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P. ion exchange chromatography after renaturation by dilution. To solve this problem, a novel purification and refolding strategy was adopted. Inclusion bodies was first purified by Q Sepharose H.P. ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200. Renatured fusion protein was obtained in a purity of more than 95%. It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80%. By the whole procedure, refolding and purification of recombinant protein can be performed within one day. This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E. coli.

Vaccination of rhesus monkeys with recombinant antigen fragments and protection from hepatitis E virus infection / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta).</p><p><b>METHODS</b>Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR.</p><p><b>RESULTS</b>Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus.</p><p><b>CONCLUSIONS</b>The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.</p>

DNA Immunization of Hepatitis E Virus (HEV) / 中国病毒学

Two plasmid constructs, pcE2 and pcE3, containing 3' fragment of open reading frame 2 (ORF2,1163 bp) of hepatitis E virus (HEV) and full-length ORF3 (369 bp), were injected into bilateral tibialis of Swiss mice respectively，for three times (0, 2nd and 4th weeks) and observed the HEV IgG by ELISA. HEV IgG was induced after the injection of pcE2 or pcE3 or both, and the percentage of seraconversion was 100% after two weeks of the third injection. Compared with injection of either construct, the antibody titers were higher in the group with combined injection of two constructs.