Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity

Objective To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A ge ne encoding rhoptry protein 1 (ROP1) from Toxoplasma gondii (T. gondii) was cloned into vector pcDNA3.Methods Amplifyied gene fragments coding for ROP1 from the genomic DNA of T.gondii ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice wer e injected at a dosage of 100 μg recombinant plasmid DNA by intramuscular inje ction and boosted after 2 weeks. pcDNA3 and normal saline were used as control . 30, 50 and 70 days after the second immunization, NK cell activity, T lympho cyte proliferation and sub-clusters and serum IgG antibody were assayed.Results The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinan t was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T ly mphocytes seen on MTT assay were higher in pcROP1 group than in the controls. T he number of CD4(+) T cells exhibited no obvious increase compared with that of the control, but CD8(+) T cells were obviously increased (P<0.05). At 90 d ays after vaccination, the titer of IgG antibody in the serum of vaccinated mice was positive (1∶100).Conclusion pcROP1 was constructed and it could elicit both cellular and humoral immune r esponses in immunized mice.     

一种新的弓形虫速殖子纯化方法

我们试用Ficoll-Urografin密度梯度离心法取得高纯度、高收获率的速殖子,现将此法报告如下。一、材料与方法 (一)弓形虫虫株 RH、ZS_1、ZS_2株为本实验室长期保种传代虫株;CN株为浙江省寄生虫病研究所引种;SH-1株采自上海第二医科大学寄生虫学教研室。5种虫株分别腹腔接种BALB/C小鼠,3d后抽取腹腔液15ml,磷酸缓冲加糖溶液洗涤3次,1000r×8min,制成速殖子悬液。 (二)Ficoll-Urografin密度梯度离心吸取5ml速殖子悬浮液加入10ml离心管内,用弯头吸管从悬浮液底部加入Ficoll-Urografin液5ml,经离心后管中形成     

登革病毒(NGC株)C与prM全长基因的扩增与克隆

目的　从登革病毒 (NGC株 )中快速分离基因组RNA ,RT -PCR扩增及克隆C和 prM全长基因 ,从而为研究蚊虫细胞对登革病毒的细胞内免疫研究奠定基础。方法　以Trizol试剂从C6 / 36细胞培养上清中提取基因组RNA ,RT -PCR扩增全长C和 prM基因 ,T -A重组入pGEM -T载体 ,重组质粒的双酶切、PCR与测序鉴定。 结果与结论　应用Trizol试剂从蚊细胞培养液上清中快速分离到高纯度和完整登革病毒RNA基因组 ,成功扩增与克隆出全长登革病毒C和 prM基因     

弓形体RH株抗原基因SAG1的扩增克隆及鉴定

目的：体外扩增弓形体主要表面抗原SAG1基因,构建pcDNA3-SAG1真核表达质粒,为研制弓形体疫苗奠定基础。方法：采用PCR技术,自行设计一对寡核酸引物（P1,P2）,从弓形体RH株基因组DNA中特异扩增出编码SAG1抗原的基因片段,扩增的目的片段经纯化后用EcoR1和HindⅢ双酶切后,克隆到真核表达质粒pcDNA3中,转化入大肠杆菌TG1,用氨苄青霉素和PCR初筛,将PCR扩增阳性的重组子分别用Sac1单酶切、EcoR1和HindⅢ双酶切鉴定,结果成功地构建真核型表达质粒pcDNA3-SAG1,从而为进一步SAG1重组抗原的体外表达创造有利条件。结果：从RH株基因组DNA中特异扩增出编码SAG1的全基因序列,扩增目的基因片段大小与预期长度（1025bp),相符;将分离、纯化的目的基因片段SAG1任入pcDNA3质粒HindⅢt和EcoR1位点,构建重组质粒pcDNA3-SAG1。结论：成功构建重组质粒pcDNA3-SAG1。     

弓形虫核酸（DNA）免疫系列研究

本系列研究筛选了弓形虫（Toxoplasma）速殖子表膜主要抗原（SAG1或P30）及分泌排泄抗原棒状体蛋白基因（ROPl），并将二者拼接构建复合基因（sAG1／ROP1），对其进行扩增、克隆、表达，制备真核表达质粒，接种小鼠，研究其免疫保护效应，为弓形虫核酸疫苗的研制提供有积极意义的科学根据。     

Immunity induced by DNA vaccine of plasmid encoding the rhoptry protein 1 gene combined with the genetic adjuvant of pcIFN-γ against Toxoplasma gondii in mice

Objective To construct the eukaryotic expression recombinant plasmid, pcIFN-γ, as a gene tic adjuvant and observe the immune responses elicited by pcDNA3-rhoptry protei n 1 （pc-ROP1） combined with pcIFN-γ against Toxoplasma gondii (T.go ndii) infection in mice.Methods A fragment of the IFN-γ gene was directly inserted into the pcDNA3 plasmid and identified by two restriction endonucleases digestion.pcIFN and pcROP1 DNA wa s injected into the left leg muscle of mice at a dosage of 100 μg, and a boost er vaccination was given at the same dosage after two weeks.Control groups wer e injected with pcDNA3 blank plasmid or normal saline.At 30, 50 and 70 days af ter booster injection, kinetic tests were carried out: MTT assay for the prolife ration response of T lymphocyte cells and the activity of NK cells, sandwich ABC -ELISA for the determination of IFN-γ, IL-2 and IL-10; a serum enzymetic aa ssay for nitric oxide (NO) in sera and ELISA for the titer of IgG antibody in sera.Results The recombinant plasmid, pcIFN-γ was constructed.The proliferation response of spleen T lymph cells, NK cell killing activity, and serum levels of IFN- γ, IL-2 and NO in mice injected with pcROP1 and pcIFN-γ were higher than in those injected with pcROP1 alone.There was no difference in IgG antibody level s between the two groups. Conclusion The genetic adjuvant, pcIFN-γ, could enhance the cellular immune response indu ced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection.     

含弓形虫ROP1基因真核表达重组质粒DNA免疫小鼠的研究Ⅴ.IFN-γ基因真核表达重组质粒的构建及其在DNA免疫中基因佐剂的作用

目的构建IFN-γ基因真核表达重组质粒作为基因佐剂,观察其与pcDNA3-ROP1(pc-ROP1) DNA共同免疫小鼠所诱导的免疫应答.方法将IF N-γ基因片段定向插入真核表达载体pcDNA3,双酶切鉴定,获得pcIFN重组子;碱裂解法大量制备,经肌肉注射免疫BALB/c小鼠,每只鼠注射pcIFN、pc-ROP1各100μg,两周后同量加强免疫一次,以pcDNA3空质粒及生理盐水组为对照.分别于免疫后第30天、50天、70 天共三次用MTT法测定小鼠脾脏T淋巴细胞增殖活性及NK细胞活性;双夹心ELISA测定血清细胞因子IFN-γ、IL-2及酶法测定NO含量;ELISA法测定IgG抗体滴度.结果构建成功的作用下,该两项指标均明显提高,且IFN-γ、IL-2及NO水平均较不加佐剂组显著提高(P＜0.01);而对IgG抗体滴度无显著影响(P＞0.05).结论 IFN-γ基因佐剂具有协同pc-ROP1DNA免疫的作用,可增强免疫鼠细胞免疫应答,IFN-γ、IL-2细胞因子及NO的产生.     

庆贺《广东寄生虫学会年报》创刊22周年暨改为《热带医学杂志》

《广东寄生虫学会年报》于1979年创刊,至今已历22年.《年报》编辑出版的22年历程中,它对我省寄生虫学和寄生虫病学术交流、促进学科的发展和防治寄生虫病起到了积极的推动作用.1978年恢复广东省寄生虫学会活动后,1979年在我国著名寄生虫学家徐秉馄教授主持下创办了《广东寄生虫学会年报》,并担任主编,朱师晦教授和潘世定所长任副主编,江静波教授等10多位教授任编委,经10年时间共出版了10卷.徐教授之后,《年报》继承了其办刊的科学性和严谨性,确保刊物的学术水平,继续出版了12卷,先后由何桂铭教授、     

我国人体弓形体感染

据估计世界弓形体感染率为25～50％左右,我国从50年代以来已从兔、猫、鼠及猪等动物分离到弓形虫。但我国人体病例的报道仍很少,现将我国人体弓形体病例和血清学调查情况作一简介。弓形体病例的发现和病原体分离至目前为止,我国至少已有7例弓形体病,其中3例由我们鉴定诊