目的 构建编码免疫佐剂Lipo多肽与登革病毒1-4型包膜蛋白EDⅢ区的重组甲病毒载体,并在小鼠体内研究其免疫效应,为研制新型的登革病毒四价疫苗奠定基础.方法 先通过GS linker将编码免疫佐剂Lipo多肽的基因序列与编码登革病毒1-4型包膜蛋白EDⅢ区的基因序列进行串联获得LipoEDⅢ;然后将LipoEDⅢ基因插入甲病毒载体DREP的多克隆位点,构建重组甲病毒载体DREP-LipoEDⅢ;将DREP-LipoEDⅢ转染293细胞,Western blot检测融合蛋白的表达;随后将DREP LipoEDⅢ免疫ICR小鼠,不加任何佐剂,再加强免疫2次后,通过ELISA检测血清中的抗体效价和细胞因子的分泌情况来评价其免疫效应.结果 成功构建了含有“融合免疫佐剂Lipo多肽与包膜蛋白EDⅢ区”的重组甲病毒质粒DREP-LipoEDⅢ.DREP-LipoEDⅢ免疫小鼠后可以诱导产生特异性抗体,抗体效价为1∶160.抗原刺激小鼠脾淋巴细胞可以产生IFN r、IL 4、IL 10细胞因子,实验组的浓度明显高于对照组.结论 在不需要加入任何外源佐剂的情况下,本研究构建的融合免疫佐剂Lipo多肽的登革四价疫苗能够诱导小鼠产生特异性的体液和细胞免疫应答,且以细胞免疫应答为主,为今后新型登革病毒四价疫苗的研究奠定了基础.

Objective: To construct dengue virus envelope protein EDIII tetravalent recombinant vaccines and to analyze its immunogenicity.Methods: Firstly, the gene sequences encoding dengue virus type 1-4 enve-lope protein EDIII were analyzed by bioinformatics methods, and the representative DENV1-4 EDIII was select-ed and connected by GS linker to obtain chimeric EDIII; Then, the chimeric gene coding EDIII was inserted into the multiple cloning site of the expression vector pColdIII to constructe the recombinant vector pColdIII-EDIII. The constructed pColdIII-EDIII plasmid was transformed into E.coli BL21 cells and induced by IPTG. The re-combinant protein EDIII was puriifed by Ni-NTA afifnity chromatography and conifrmed by SDS-PAGE and Western blot analysis. The puriifed protein was used to immune BABL/c mice. After boosting twice, to detect the antibody titers in serum and the secretion of cytokines by ELISA to evaluate the immune response.Results:The mice immunized with EDIII could induce speciifc antibodies and the antibody titers was 1:40 000. The mouse spleen lymphocytes stimulated by antigen could produce cytokine including IFN-r, IL-4, IL-10, which were sig-niifcantly higher in the experimental group.Conclusion: Our studies indicates that dengue virus envelope protein EDIII tetravalent vaccine can induce speciifc humoral and cellular immune response, and give priority to with the humoral immune response, which laid the foundation for the research of a new type of chimeric tetravalent vac-cine in the future.

目的 构建表达弓形虫表面抗原SAG1的自杀性DNA疫苗pDREP-SAG1并检测其免疫原性.方法 以弓形虫RH株基因组DNA为模板,PCR扩增SAG1基因(GenBank No.HM776940.1),克隆至甲病毒复制子载体pDREP-eGFP中替换原有的eGFP基因,菌液PCR、双酶切及测序鉴定重组质粒.重组的自杀性DNA疫苗pDREP-SAG1经股四头肌注射并联合瞬时电穿孔免疫BALB/c小鼠,同时以原质粒pDREP-eGFP作为对照组,间隔3周后以相同条件再免疫1次,收集小鼠血清,以弓形虫裂解抗原Western blotting检测其诱导产生的特异性抗体.结果 从弓形虫基因组中PCR扩增得到1 011 bp长度的SAG1基因,成功构建重组质粒pDREP-SAG1.Western blotting结果显示,重组质粒免疫的小鼠血清能特异性地识别虫体抗原中的SAG1抗原.结论 成功构建了弓形虫自杀性DNA疫苗pDREP-SAG1,能诱导小鼠产生特异性的抗体,具有免疫原性.

Objective This study reviewed the approach of general practice teacher training in Zhejiang Province, evaluated the training effect and summarized training experience. It aimed to put forward recommendations for future training and teaching staff improvement. Methods There is an urgent need to increase excellent teaching staff of general practice,in order to promote the training system of general practitioner. And a training approach is designed to improve trainees' comprehensive ability. We recruited 218 trainees who received the whole training and assigned them to complete questionnaires. Their satisfaction degree about the training and ability improvement after the training were investigated. Results Among 218 subjects,95. 4%(208/218)were satisfied with the training teachers;93. 5%(204/218) were satisfied with organization and management;93. 1%(203/218) were satisfied with the rotation arrangement;69. 3%(151/218) were satisfied with the textbook;and 94. 0%(205/218) were satisfied with the overall effect. The trainees who had improvement in fundamental understanding of general practice,clinical competence and teaching ability accounted for 95. 4%(208/218),94. 9%(207/218) and 93. 5%(204/218)respectively. Conclusion Most trainees thought the expected effect of the training was achieved,and their teaching and professional ability were improved after the training. Problems still exist,including deficiencies in the textbook,the lack of time of receiving training due to busy work,and the lack of benefit and allowance during the training.

因住院医师规范化培训实施过程中发现带教师资在带教方面普遍存在着一定的问题,影响了住院医师规范化培训质量,亟需实施师资培训.在住院医师规范化培训师资的带教特性基础上,以岗位胜任力模型为抓手,分析浙江省住院医师规范化培训师资课程的设计.

To clone and express autophagy-related protein 3 (TgAtg3) gene of Toxoplasma gondii, and obtain the specific polyclonal antibody against TgAtg3.TgAtg3 cDNA was inserted into prokaryotic expression vector pET28a. After identification, the constructed plasmid pET28a-TgAtg3 was transformed into E. coli Rosetta cells, and induced by special induction medium for expression of the protein. The recombinant protein was purified via Ni-NTA affinity chromatography. Western blotting assay was performed with anti-His tag mouse monoclonal antibody as the primary antibody. Rabbits were immunized with 125 μg purified TgAtg recombinant protein. Each rabbit received 4 immunizations at 2-week intervals with the same dose of antigen. The specific anti-TgAtg3 polyclonal antibody was obtained, and analyzed by Western blotting and indirect immunofluorescence assay (IFA).pET28a-TgAtg3 plasmid was identified by restriction enzyme digestion, PCR amplification and sequencing. SDS-PAGE and Western blotting analysis showed that the recombinant TgAtg3 protein (about Mr 44,000) was expressed in E. coli Rosetta cells. TgAtg3 protein from tachyzoite lysates was recognized by the specific anti-TgAtg3 polyclonal antibody. IFA assay determined that the specific polyclonal antibody bound to TgAtg3 protein from the cytoplasm of tachyzoites.The obtained soluble polyclonal antibody against TgAtg3 can specifically react to the endogenous TgAtg3 protein.

目的 探讨左氧氟沙星治疗社区获得性肺炎(CAP)的临床疗效.方法 将152例CAP患者随机分为观察组78例和对照组74例,分别给予左氧氟沙星和阿奇霉素序贯疗法治疗,观察两组患者疗效.结果 观察组临床症状及体征改善时间、临床疗效及细菌清除率均优于对照组(P＜0.05).结论 左氧氟沙星序贯疗法治疗CAP具有疗效好、见效快、抗菌作用强等优点,且安全性较好,值得临床借鉴采用.

Angiostrongylus cantonensis(Ac)is an important pathogen of causing eosinophilic meningitis or meningoencephalitis.The larvae invade and migrate in human body,then invade the central nervous system causing Angiostrongyliasis cantonensis.But the prevention and treatment of the disease are still relatively limited.The study of functional proteins is expected to discuss the disease occurrence and development mechanism at cellular and molecular level.It is also helpful to the diagnosis and the development of new drugs.In this paper,we reviewed the progress of study on functional proteins of Angiostrongylus cantonensis.

Objective To elucidate the Liushi chronic obstructive pulmonary disease(COPD) to investigate the prevalence of epidemiology,age trends and analysis of the related risk factors and provide scientific basis for the prevention and control work effectively in the future.Methods In 2008 December-2012 year in October,by outpatients and inpatients were registered in 3108,inquiry(including living conditions,living and cooking habits,smoking history,family history and past history).Physical examination,complete epidemiological survey of pulmonary function test,and make corresponding electrocardiogram and chest CT ray examination,risk factors for COPD were analyzed by univariate and multivariate unconditional logistic regression analysis.Results The total prevalence rate of COPD was 13.51%,gender,age,smoking history,cough in childhood and other factors in the COPD group compared with the non-COPD group had significant difference.The area the incidence of COPD accounted for 40 of the population aged above 1.73%.Conclusion COPD in the area with higher prevalence,gender,age,smoking,history of childhood cough is closely related with COPD,and the interior and exterior environment and occupation exposure factors further research of many COPD complications,should be emphasized.

To establish and optimize two-dimensional gel electrophoresis(2-DE) models for proteomic analysis of A.cantonensis larvaeⅤ,the protein of larvae Ⅴ of A.cantonensis was extracted by grinding and repeated freezing and thawing,then 2-DE was carried out respectively with the strip of pH3-10 and pH4-7 after measuring the concentration.The gel electrophoresis image was compared after silver staining.The results indicated that the protein spots were more concentrated,and the space among spots were smaller with IPG strip of pH3-10.Then we obtained high-resolute,uniform distributed protein spots,719 protein spots with IPG strip of pH4-7,and the space among spots were relatively large,avoiding to protein spots of high abundance cover the low abundance,it has higher sensitivity.As a result,establishment and optimization of 2-DE models for A.cantonensis was successfully conducted,and 2-DE gel with high resolution was obtained,which might provide foundation for the proteomic analysis of A.cantonensis.

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Toxoplasma gondii DNA in mice infected with T. gondii PrU strain.this lAMP assay was based on the sequence of highly repetitive B1 gene.the detection limit of T. gondii lAMP assay was 1 pg of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites.The LAMP assay was also highly specific for T. gondii and able to detect T. gondii DNA in urine of mice treated with dexamethasone at 90 day post infection (p.i.), although this assay could not detect the DNA in mice urine 2-6 days p.i. these results demonstrated that lAMP is effective for evaluation of therapy effectiveness for T. gondii infection.the established lAMP assay may represent a useful and practical tool for the routine diagnosis and therapeutic evaluation of human toxoplasmosis.

Aeromonas hydrophila was recognized as a human pathogen for decades.However,it still remains under development for an effective vaccine against this bacterium.The aim of this study was to investigate the impact of PEG as an ultrasonic carrier and vaccine adjuvant on the immune protection of vaccine against A.hydrophila.Cryprinuscarpiod was injected intraperitioneally with control vaccine and PEG-conjugated vaccine,respectively.The percentage and index of phagocytosis,antibacterial activities,antibody titer and the relative survival percentage were thereafter examined.The results showed that all the immune response indicators of PEG6000-conjugated vaccine were significantly higher than those of control vaccine.Particularly,the relative survival percentage of PEG6000-conjugated vaccine reached 77.8%,which was 22.2% higher than that of inactivated control vaccine.Our results indicate that PEG6000 is a highly effective ultrasonic carrier and adjuvant in the preparation of vaccine against A.hydrophila.

Polyethylene glycol(PEG)-conjugated vaccine of Vibrio harveyi was made by PEG which was used for ultrasonic carrier and ultrasonic medium,and by the inactivation methods of transient high-temperature,high press and shearing force of ultrasonic.For comparing the difference between two kinds of vaccine,the PEG-conjugated vaccine and traditional vaccine were injected into Cryprinuscarpiod intraperitioneally,respectively,then the percentage of phagocytosis,the index of phagocytosis,antibacterial activities,antibody titer and relative percent survival were checked.The results showed all the indicators and absolute value of PEG group were significantly higher than other experimental groups.And the relative percent survival was 89.5% for PEG 6000 vaccine,which is the highest,and it was 31.6% higher than that of traditional inactivated group.PEG 6000 vaccine could provide the best protection for Cryprinuscarpiod against Vibrio harveyi.

The effect of perfluorooctane sulphonate(PFOS) on the Vibrio harveyi cell density and expression of extra-toxin gene was investigated.The Vibrio harveyi samples,which cultured in LB media with different PFOS concentrations(5,50,500 mmol/L),were collected by centrifugation(4 000 r/min) after 6,15 and 24 h incubation,respectively,and cell sediments were re-suspend in phosphate buffer solution(PBS).And the RT-PCR was used for analyzing the gene expression of extracellular protease,hemolysin and 16 SrRNA conserved sequence.The results shown that the cell density was inhibited significantly in the medium with 5 mmol/L PFOS(P0.01),but not for 50 mmol/L and 500 mmol/L.While three different concentration of PFOS enhanced the expression gene of ECP and hemolysin(P0.01).However,the gene expression of 16 SrRNA conserved sequence was boosted by 50 mmol/L PFOS only(P0.01).The hemolysin was also sensitive to the lower PFOS concentration.In sum,the data in this paper indicated the lower PFOS concentration will impact on the growth and gene expression of Vibrio harveyi.

To understand the pollution of shellfish due to Listeria monocytogenes(Lm)in Wenzhou city.Result showed that 3 samples out of 100 samples was found,which accounted for 3.00%.The shellfish in Wenzhou city had been polluted by Lm,the sanitary inspection of which should be more strengthened.

PEG conjugate vaccine of Aeromonas hydrophila was made by the methods of transient high-temperature,high press and shearing force of ultrasonic,using PEG as ultrasonic medium and vaccine vector.The PEG conjugate vaccine and the traditional vaccine were injected Cryprinuscarpiod intraperitioneally respectively.The percentage of phagocytosis,the index of phagocytosis,antibacterial activity,antibody titer and immune protective efficiency were investigated.The results showed that all the testing indexes of PEG conjugate vaccine were significantly higher than other vaccines.The immune protective efficiency was 80% for PEG conjugate vaccine,which was the highest and 26.7% higher than that of traditional vaccine,indicating that PEG6000 was a good binder of vaccine preparation for Cryprinuscarpiod against Aeromonas hydrophila.

Objective A specific candidate antigen of stage V larvae of Angiostrongylus cantonensis was cloned,expressed,and identified.Methods Based on the results of screening the stage V larvae of A.cantonensis cDNA library using immuno-probes,the Ac11 gene was immunoreactive.The Ac11 gene was amplified and submitted for BLAST analysis online.Then,it was subcloned into the expression vector pET28a(+) and expressed in E.coli.The recombinant protein Ac11 was purified and identified immunologically.Results The Ac11 gene was predicted to encode sulfite oxidase.The recombinant plasmid pET28a(+)-Ac11 was successfully constructed.The recombinant protein Ac11 with a molecular weight of about 30 kd was highly expressed in the form of inclusion bodies according to SDS-PAGE analysis.It was also highly immunoreactive in Western blot.Conclusion The recombinant protein Ac11,which is highly immunoreactive,was obtained in vitro and may provide a basis for further immunodiagnostic study of angiostrongyliasis.

OBJECTIVE: To investigate the status of bacterial contamination in the shellfish products in Wenzhou. METHODS: One hundred samples were collected and their bacterial populations including the total plate count were investigated. RESULTS: Of the 100 samples collected, 67 samples failed to not meet the national regulations due to bacterial contamination, accounting for 67% of the total samples. Among the contaminated samples, the most serious contamination was caused by coliforms (61.4% of the total plate count with contamination), followed by Salmonella (18.6%), Vibio parahaemolyticus (15.7%), Listeria spp. (4.3%) and others (6%). CONCLUSION: Microbial pollution has become a threat to the marine shellfish products in Wenzhou.

Objective To construct and screen a cDNA library of Ⅴ stage larvae of Angiostrongylus cantonensis to ob-tain specific antigen genes for diagnosis. Methods Total RNA was extracted from Ⅴ stage larvae of A. Cantonensis.After that, an unamplified library was constructed the using SMART~(TM) cDNA Library Construction Kit from Clontech Laboratories. The titer and the recombination rate of the unamplified library were detected and then amplified. Positive clones were obtained by using antisera of rats infected with A. Cantonensis as an immuno-probe. Some of the inserts of positive clones were specifically amplified by PCR and sequenced. The nucleotides and the prediction of open reading frames (ORFs) and physico-chemical properties of deduced amino acids were analyzed using bioinformatics. Results This study constructed a cDNA library of Ⅴ stage larvae of A. Cantonensis using SMART LD-PCR, and eleven positive clones were obtained through preliminary immunoscreening. The inserts of nine positive clones were sequenced and ana-lyzed for their homology in the GenBank database. Results showed that one EST of Ⅴ stage larvae of A. Cantonensis had homology of 99% and that one was not homologous to genes in GenBank. The maximum homology of the other six se-quences, two of which were the same, was 91% in comparison to nucleotides from Caenorhabditis elegans and Cae-norhabditis briggsae. There were total seven positive clones with complete ORFs. Conclusion Constructing and immu-noscreening a cDNA library of Ⅴ stage larvae of A. Cantonensis yielded seven significant clones, providing a basis for fu-ture research on these genes.

To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium,the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly.The positive clones were identified by EcoR I,Xho I digestion and sequenced.The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares.The P30 gene was recombined with plamid pET-28 a(+) to construct the prokaryotic expression vector and was expressed in E.coli with IPTG induction.Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting.It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified,and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively.Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes.The expressed plasmid was identified by EcoR I/Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay.It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.