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Chinese Medical Journal 2001年第3期114卷 93-96,111页

作者：郭虹陈观今吕芳丽陈海峰郑焕钦中山医科大学寄生虫学研究所广州510089

Objective To construct the eukaryotic expression recombinant plasmid, pcIFN γ, as a genetic adjuvant and observe the immune responses elicited by pcDNA3 rhoptry protein 1 (pc ROP1) combined with pcIFN γ against Toxoplasma gondii (T gondii) 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 was injected into the left leg muscle of mice at a dosage of 100?μg, and a booster vaccination was given at the same dosage after two weeks Control groups were injected with pcDNA3 blank plasmid or normal saline At 30, 50 and 70 days after booster injection, kinetic tests were carried out: MTT assay for the proliferation 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 aassay 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 levels between the two groups Conclusion The genetic adjuvant, pcIFN γ, could enhance the cellular immune response induced by DNA vaccine of pcROP1 in mice against Toxoplasma gondii infection

关键词： Toxoplasma gondii · rhoptry protein 1 · DNA vaccination · genetic adjuvant · pcIFN γ

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

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Chinese Medical Journal 2001年第8期114卷 54-57,107页

作者：陈观今郭虹吕芳丽郑焕钦中山医科大学寄生虫学研究所广州510089

Objective　To observe the immune responses elicited in BALB/c mice by a DNA vaccine. A gene 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 *** ZS2 were inserted into cloning vector, pUC18, and sub-cloned into pcDNA3. Mice were injected at a dosage of 100?μg recombinant plasmid DNA by intramuscular injection 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 lymphocyte proliferation and sub-clusters and serum IgG antibody were ***　The specific gene fragment coding for ROP1 was amplified and a pcROP1 recombinant was constructed. At 30 days after immunization, the spleens of the mice were obviously enlarged evidently. NKC activity and the proliferation of spleen T lymphocytes seen on MTT assay were higher in pcROP1 group than in the controls. The 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 days 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 responses in immunized mice.

关键词： Toxoplasma gondii · rhoptry protein 1 · pcROP1 recombinant plasmid · cloning · DNA immunity

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微茎类吸虫阴茎及生殖孔的扫描电镜观察

Current Zoology 1987年第4期33卷 381-382页

作者：余新炳柯小麟中山医科大学寄生虫学研究所中山医科大学寄生虫学研究所广州

在吸虫分类学上,生殖系统的具体构造是其主要的分类特征之一。在雌雄同体的吸虫,阴茎及生殖孔构造的差异亦造成机械性的种间隔离,形成新种,对徽茎类吸虫,阴茎的形态特征在其分类学上占有相当重要的地位。但迄今人们所观察到吸虫的阴茎... 详细信息

在吸虫分类学上,生殖系统的具体构造是其主要的分类特征之一。在雌雄同体的吸虫,阴茎及生殖孔构造的差异亦造成机械性的种间隔离,形成新种,对徽茎类吸虫,阴茎的形态特征在其分类学上占有相当重要的地位。但迄今人们所观察到吸虫的阴茎及生殖孔的形态基本上都是光学显微镜下展示的结构,在电子显微镜下对其精细构造的具体研究工作尚未见报道。本文就对10种微茎类吸虫的阴茎及生殖孔在扫描电镜下所观察到的结果作一简报。

关键词：吸虫雌雄同体蠕虫阴茎男生殖器生殖孔微茎

弓形虫ROP1基因真核表达重组质粒免疫小鼠后的免疫应答

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中国寄生虫学与寄生虫病杂志 1999年第6期17卷 334-337页

作者：郭虹陈观今郑焕钦周永安吕芳丽中山医科大学寄生虫学研究所广州510089

目的：用构建的弓形虫ｐｃＤＮＡ３－ＲＯＰ１真核表达重组质粒，直接免疫小鼠，观察其所诱导的细胞及体液免疫反应。方法：碱裂解法大量制备ｐｃＤＮＡ３－ＲＯＰ１质粒，经肌肉注射免疫ＢＡＬＢ／ｃ小鼠，每鼠注射１００ μ... 详细信息

目的：用构建的弓形虫ｐｃＤＮＡ３－ＲＯＰ１真核表达重组质粒，直接免疫小鼠，观察其所诱导的细胞及体液免疫反应。方法：碱裂解法大量制备ｐｃＤＮＡ３－ＲＯＰ１质粒，经肌肉注射免疫ＢＡＬＢ／ｃ小鼠，每鼠注射１００ μｇ，２ｗｋ后同量加强免疫１次，以ｐｃＤＮＡ３空质粒及生理盐水组为对照。分别于免疫后３０ｄ、５０ｄ、７０ｄ共３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；用间接免疫荧光抗体法测定Ｔ淋巴细胞亚群数目；ＥＬＩＳＡ法测定ＩｇＧ抗体滴度。结果：用ｐｃＤＮＡ３－ＲＯＰ１质粒免疫小鼠３０ｄ后，脾脏明显增大；免疫组脾淋巴细胞增殖活性明显高于生理盐水及空质粒对照组。ＮＫ细胞杀伤活性３次的测定结果免疫组均高于对照组。Ｔ细胞亚群，ＣＤ４＋细胞数与对照组相比较无明显变化，而ＣＤ８＋细胞数显著增高。血清抗体ＩｇＧ７０ｄ内检测结果，与对照组相比较，无明显增高；而免疫后９０ｄ检测明显增高，抗体滴度１∶１００。结论：用ｐｃＤＮＡ３－ＲＯＰ１重组质粒ＤＮＡ免疫小鼠，可诱导产生细胞及体液免疫应答。

关键词：弓形虫棒状体蛋白1 重组质粒 DNA 免疫应答

恶性疟原虫FCC1/HN株CSP抗原基因表达产物在小鼠体内的免疫应答研究

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中国寄生虫学与寄生虫病杂志 1999年第6期17卷 325-329页

作者：方政余新炳刘彦文罗树红中山医科大学寄生虫学研究所

目的：利用ｐｃＤＮＡ３质粒作为载体，在人宫颈癌细胞（ＨｅＬａ细胞）中高效表达恶性疟原虫环子孢子蛋白（ＣＳＰ），观察表达产物诱导ＢＡＬＢ／ｃ小鼠免疫的应答水平。方法：目的基因ＰｆＣＳＰ在ＨｅＬａ细胞中表达，将... 详细信息

目的：利用ｐｃＤＮＡ３质粒作为载体，在人宫颈癌细胞（ＨｅＬａ细胞）中高效表达恶性疟原虫环子孢子蛋白（ＣＳＰ），观察表达产物诱导ＢＡＬＢ／ｃ小鼠免疫的应答水平。方法：目的基因ＰｆＣＳＰ在ＨｅＬａ细胞中表达，将纯化的表达产物免疫接种小鼠，通过ＥＬＩＳＡ、Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ分析、Ｔ淋巴细胞增殖实验、ＮＫ细胞活性检测和Ｔ淋巴细胞亚群测定，观察其诱导ＢＡＬＢ／ｃ小鼠产生体液免疫和细胞免疫的应答水平。结果：ＥＬＩＳＡ检测抗体滴度达１∶６４００；Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ结果在３８．３ｋＤａ相应位置出现较清晰的显色条带；表达产物能特异刺激小鼠脾淋巴细胞增殖、ＣＤ４＋与ＣＤ８＋细胞有所增加，并能提高小鼠ＮＫ细胞活性。结论：真核表达系统ｐｃＤＮＡ３－Ｐｆ／ＨｅＬａ表达产物能特异刺激小鼠产生体液免疫和细胞免疫的应答，

关键词：恶性疟原虫环子孢子蛋白 HeLa细胞表达产物

紫外线减毒弓形虫ZS1株滋养体在小鼠体内的细胞免疫反应

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中国寄生虫学与寄生虫病杂志 1999年第6期17卷 384-386页

作者：吕芳丽郑焕钦郭虹陈观今中山医科大学寄生虫学研究所广州510089

目的：探讨紫外线减毒弓形虫ＺＳ１株在小鼠体内的免疫保护性和细胞免疫反应。方法：用波长为２５３７°Ａ的紫外线照射弓形虫ＺＳ１株滋养体，照射高度为５ｃｍ，照射时间６０ｍｉｎ。小鼠于免疫后４５ｄ用同株滋养体攻击... 详细信息

目的：探讨紫外线减毒弓形虫ＺＳ１株在小鼠体内的免疫保护性和细胞免疫反应。方法：用波长为２５３７°Ａ的紫外线照射弓形虫ＺＳ１株滋养体，照射高度为５ｃｍ，照射时间６０ｍｉｎ。小鼠于免疫后４５ｄ用同株滋养体攻击感染，攻击后４ｄ剖杀，与单免疫组、单感染组及正常对照组小鼠比较其脾Ｔ淋巴细胞增殖反应及其亚群的变化。结果：小鼠接种紫外线减毒弓形虫ＺＳ１株滋养体后能正常存活，于接种后４９ｄ各组织未查见滋养体、包囊或假包囊；免疫组攻击感染后存活时间较单感染组延长；体外特异抗原刺激后，可诱导免疫组及免疫攻击组强的脾Ｔ淋巴细胞增殖反应；免疫攻击组ＣＤ４＋Ｔ细胞明显下降，ＣＤ４＋／ＣＤ８＋比率倒置；免疫组、免疫攻击组及感染组的ＮＫ细胞活性均明显增强。结论：紫外线减毒弓形虫ＺＳ１株滋养体疫苗能够诱导免疫小鼠产生一定的抗攻击感染保护力，其中ＣＤ８＋Ｔ细胞和ＮＫ细胞可能发挥着重要作用。

关键词：弓形虫紫外线减毒细胞免疫反应

Immune responses to the expressed products of the CSP antigen gene of Plasmodi um falciparum southern China isolate FCC1/HN in Hela cell

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Chinese Medical Journal 2001年第8期114卷 61-64,108页

作者：方政余新炳刘彦文罗树红南通医学院寄生虫学教研室南通226001 中山医科大学寄生虫学研究所广州510089

Objective　To construct a eukaryotic expression system with pcDNA3-PfCSP/Hela for the Circumsporozoite protein (CSP) gene of Plasmodium falciparum (P. falciparum), to observe the immune responses in BALB/c mice induced by the expressed proteins. Methods　The recombinant plasmid pcDNA3-PfCSP was transformed into the Hela cell line. The expressed protein was isolated and analyzed by using SDS-PAGE and used for immunization of BALB/c mice by subcutaneous, intravenous, and intraperitoneal adminstration. Enzyme-linked immunosorbent assay(ELISA), Dot-ELISA, Western blot, T lymphocyte proliferation test, natural killer cell(NKC) activity assay, and CD4+ and CD8+ T cell detection were used for observation of humoral and cellular immune responses. Results　Immune sera strongly reacted with the expressed protein, antibody titer was up to 1∶6400 as detected by ELISA. Western blot analysis revealed a specific band at 38.3?Kda. When the spleen cells of normal and immunized BALB/c mice were specifically stimulated with expressed protein, the optical densities were 0.12±0.03 and 0.34±0.04, respectively. The latter were significantly higher than the former (P<0.01). We used the MTT colorimetric assay to measure NKC activity of mice spleen. The results showed that the NKC activity of immunized BALB/c mice was remarkably higher than that of the controls (P<0.05). CD4+ and CD8+ T cells were detected by using monoclonal antibody immunofluorescence methods. The results showed that the percentage of CD4+ and CD8+ T cells of immunized group were significantly higher than that of control group (P<0.05).Conclusions　The humoral and cell-mediated immune responses and elevated NKC activity to products made with a eukaryotic expression system could be specifically detected in BALB/c mice. These findings indicate that the expressed protein could enhance the immune function in mice.

关键词： plasmodium falciparum · circumsporozoite protei n · Hela cell · expressed product · immune response

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日本血吸虫卵壳蛋白基因的克隆与表达

中国寄生虫学与寄生虫病杂志 1998年第6期16卷 415-420页

作者：刘兰英余新炳中山医科大学寄生虫学教研室

目的：在体外高效表达具有生物学和免疫学活性的日本血吸虫卵壳蛋白，探讨其作为抗卵免疫靶抗原的可能性。方法：采用ＰＣＲ技术扩增日本血吸虫卵壳蛋白基因，将其克隆到ｐｃＤＮＡ３质粒，在ＨｅＬａ细胞中高效表达，对表达产物进行鉴... 详细信息

目的：在体外高效表达具有生物学和免疫学活性的日本血吸虫卵壳蛋白，探讨其作为抗卵免疫靶抗原的可能性。方法：采用ＰＣＲ技术扩增日本血吸虫卵壳蛋白基因，将其克隆到ｐｃＤＮＡ３质粒，在ＨｅＬａ细胞中高效表达，对表达产物进行鉴定和免疫原性研究。结果：特异扩增日本血吸虫卵壳蛋白基因编码序列（６１８ｂｐ），构建了真核表达质粒ｐｃＤＮＡ３／ＥＳＧ，在ＨｅＬａ细胞中高效表达卵壳蛋白，其表达量高达２４．４％，分子量约为２２ｋＤａ，ｄｏｔ－ＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ分析显示，表达蛋白能与感染兔血清及虫卵免疫血清产生较强的免疫反应；此外，表达蛋白还能特异刺激经虫卵免疫后的ＢＡＬＢ／ｃ小鼠脾细胞增殖，其转化率达４４．５７％。结论：成功地构建重组质粒ｐｃＤＮＡ３／ＥＳＧ，并在ＨｅＬａ细胞中高效表达卵壳蛋白基因。

关键词：日本血吸虫卵壳蛋白克隆表达

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卡氏肺孢子虫感染裸小鼠的实验研究

中国人兽共患病杂志 1992年第5期8卷 20-21页

作者：陈发凯徐秉锟中山医科大学寄生虫学研究所

用醋酸可的松诱导Wistar大鼠获得卡氏肺孢子虫感染,取大鼠肺组织含虫匀装直接接种于NCS系裸小鼠两侧肺0.05ml,以及接种保存于-70℃低温中5月以上的含虫肺匀奖和分离纯化后的虫体,均使裸小鼠获得感染,发生卡氏肺孢子虫肺炎。在国内首先... 详细信息

用醋酸可的松诱导Wistar大鼠获得卡氏肺孢子虫感染,取大鼠肺组织含虫匀装直接接种于NCS系裸小鼠两侧肺0.05ml,以及接种保存于-70℃低温中5月以上的含虫肺匀奖和分离纯化后的虫体,均使裸小鼠获得感染,发生卡氏肺孢子虫肺炎。在国内首先建立起裸小鼠卡氏肺孢子虫肺炎实验模型。

关键词：裸小鼠孢子虫感染

弓形虫ROP1基因真核表达重组质粒DNA免疫小鼠的研究Ⅰ.pcDNA3-ROP1真核表达重组质粒的构建

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中国人兽共患病杂志 1999年第1期15卷 3-5页

作者：郭虹陈观今周永安郑焕钦刘彦文中山医科大学寄生虫学研究所

目的构建弓形虫ＺＳ２株ｐｃＤＮＡ３－ＲＯＰ１真核表达重组质粒，为进一步表达及ＤＮＡ免疫做准备。方法用ＰＣＲ技术从弓形虫ＺＳ２分离株的基因组ＤＮＡ中扩增编码棒状体蛋白１（ＲＯＰ１）的基因片段，重组入ｐＵＣ１８克隆载体。... 详细信息

目的构建弓形虫ＺＳ２株ｐｃＤＮＡ３－ＲＯＰ１真核表达重组质粒，为进一步表达及ＤＮＡ免疫做准备。方法用ＰＣＲ技术从弓形虫ＺＳ２分离株的基因组ＤＮＡ中扩增编码棒状体蛋白１（ＲＯＰ１）的基因片段，重组入ｐＵＣ１８克隆载体。将ｐＵＣ１８－ＲＯＰ１中的ＲＯＰ１外源基因片段经酶切、连接等反应，亚克隆入ｐｃＤＮＡ３真核表达载体，再经含氨苄ＬＢ培养基筛选，酶切、ＰＣＲ鉴定。结果从ＺＳ２株基因组ＤＮＡ中扩增出特异的ＲＯＰ１基因片段，克隆成功ｐＵＣ１８－ＲＯＰ１；经亚克隆，筛选鉴定获得了ｐｃＤＮＡ－ＲＯＰ１重组质粒。结论构建成功弓形虫ｐＵＣ１８－ＲＯＰ１重组质粒，亚克隆成功ｐｃＤＮＡ－ＲＯＰ１重组质粒。

关键词：克隆重组质粒弓形体病 ROP1基因 DNA免疫