Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

Hepatitis C(HCV) genome is highly variable,particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene.The variability of HCV genome has been a major obstacle for de-veloping HCV vaccines.Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes,we synthesized an minigene of HCV-derived multi-epitope peptide an-tigen(CMEP) ,which contains 9 B-cell HVR1 mimotopes in E2,2 conserved CTL epitopes in C,1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3.This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP.The immunogenic properties of CEMP were characterized by HCV infected patients' sera,and found that the reactivity frequency reached 75%.The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%.Meanwhile,we constructed an HCV DNA vaccine candidate,plasmid pVAX1.0-st-CMEP carrying the recombinant gene(st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene.Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody,which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes,and would be of the value as a candidate for the development of HCV vaccines.     

α-Galactosidase from Alfalfa Seeds (Medicago sativa L.)

An α-galactosidase from alfalfa seeds was purified 140-fold by ammonium sulfate fractionation, and column chromatography on Sephadex G-100, DEAE- and CM-Sephadex. Polyacrylamide-gel electrophoresis of the purified enzyme showed a single protein band. The molecular weight was estimated to be approximately 57,000 by gel-filtration. The purified enzyme hydrolyzed p-nitrophenyl α-d-galactoside more rapidly than raffinose. The maximal enzyme activities were obtained at pH 4.0 and 5.5 for p-nitrophenyl α-d-galactoside and at 4.5 for raffinose. The enzyme was shown to be inhibited by Hg²⁺ and Ag⁺ ions, and d-galactose.     

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166–247 aa, S1 gene; 501–515 aa, S1 gene; 8–30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD₄₅₀ value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.     

EV71多表位抗原亲和纯化及类病毒颗粒胞外自组装

利用肠道病毒71型（EV71）衣壳蛋白优势表位肽段构建融合蛋白抗原能有效抑制病毒感染,有望成为继灭活病毒后更为安全有效的疫苗品种。该融合蛋白能通过原核体系有效表达但形成无序包涵体,采用常规层析介质难以实现目标蛋白与宿主杂质的有效分离,阻碍了对该抗原蛋白进行全面临床前活性及安全性评价。在原有融合蛋白抗原N端插入组氨酸标签,对形成的包涵体变性溶解后直接采用镍金属螯合亲和介质进行分离纯化,获得了纯度大于95%的抗原纯品,目标蛋白收率46.8%。采用透析方式脱除纯化样品中高浓度脲,发现直接透析至无脲的缓冲液中蛋白质大量沉淀,而先稀释至2mol/L脲的缓冲液中然后用G25脱盐柱完全脱除脲则无任何沉淀形成,获得近100%的蛋白质收率。透射电镜分析最终样品发现融合蛋白形成了10nm左右粒径均一的类病毒蛋白颗粒,且在pH 8.0的磷酸盐缓冲液中保持稳定。该研究结果为将EV71融合蛋白抗原发展为安全有效且低成本的手足口疫苗奠定了基础。     

Activités Polyphénoloxydasique et Peroxydasique du Fruit de la Tomate (Lycopersicum esculentum) Purification et Quelques Propriétés

During isolation and purification of tomato polyphenoloxidase and peroxidase, a peroxidase activity tightly bound to the polyphenoloxidase was found at all steps of the process of purification. From these results and from the values obtained for optimum pH (6.6), isoelectric point (pH=4.0) and electrophoretic pattern of these activities, the occurence of only one enzyme is suggested. This enzyme is probably, a peroxidase able to oxidise phenolic substrates with oxygen as second substrate, as indicated by the value obtained for molecular weight (45,000±5,000).     

Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3) were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST). The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund''s adjuvant. Mouse splenocytes were then cultured in Dulbecco''s Modified Eagle''s Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL), but interleukin (IL)-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6%) was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj) ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.     

A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice

We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein （MOMP） multi-epitope of Chlamydia trachomatis. A short gene of muiti-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a（＋） containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His- MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multiepitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies （lgG1 and IgG2a）, but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi- epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Thl response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi- epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.