弓形虫表面抗原SAG1 DNA疫苗的构建

目的　构建真核重组表达质粒 pVAX1-SAG1,并探讨其诱导的体液免疫应答。 方法　采用多聚酶链反应技术 ,从弓形虫RH株基因组DNA中扩增截短型的SAG1基因片断 ,并克隆至载体pMD18-T ,经菌落PCR鉴定和测序分析后 ,亚克隆至真核表达载体 pVAX1,构建重组真核表达质粒pVAX1-SAG1,并予菌落PCR和双酶切鉴定。以此为疫苗候选分子免疫小鼠 ,检测其诱导产生的抗体。结果　PCR扩增出SAG1基因的截短型片段 ,其大小约 780bp ;测序的阳性TA克隆除两处发生同义突变外 ,其余序列与原序列一致 ;TA克隆的插入片段被亚克隆到真核表达载体 pVAX1,构建了重组表达质粒pVAX1-SAG1;该质粒诱导小鼠产生了抗弓形虫抗原的抗体。 结论　成功构建了弓形虫表面抗原SAG1的DNA疫苗。     

弓形虫表面抗原SAG1基因自杀性DNA疫苗的构建及免疫原性检测

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。     

弓形虫SAG2基因体外扩增、克隆及在耻垢分支杆菌中的表达

目的体外扩增弓形虫RH株表面抗原2(SAG2)基因,并构建大肠杆菌-分支杆菌穿梭质粒ps3000-SAG2,构建重组耻垢分支杆菌,免疫接种小鼠,通过检测免疫小鼠血清中的弓形虫抗体,证明SAG2重组蛋白在耻垢分支杆菌中有表达。方法采用聚合酶链式反应(PCR)方法,体外扩增弓形虫RH株的SAG2基因,克隆入pGEMT载体,然后构建亚克隆ps3000-SAG2大肠杆菌-分支杆菌穿梭质粒,经电转化方法,将SAG2基因的穿梭质粒转化到耻垢分支杆菌(Mycobacterium smegmatismc2155)中,用重组耻垢分支杆菌免疫小鼠,Western-blotting方法检测免疫小鼠血清中的弓形虫抗体。结果成功扩增了弓形虫的SAG2基因;正确构建穿梭质粒ps3000-SAG2,免疫印迹分析,免疫小鼠血清能所识别SAG2重组蛋白。结论耻垢分支杆菌在小鼠体内表达出了重组蛋白SAG2,具有抗原性,刺激小鼠机体产生出了抗弓形虫的抗体,为下一步弓形虫重组BCG(Bacilli Calmette-Guérin)疫苗的研究奠定了基础。     

弓形虫致密颗粒抗原GRA8的原核和真核表达质粒的构建

目的 构建弓形虫RH株致密颗粒抗原GRA8的原核和真核重组表达质粒。方法 参照GRA8序列分别设计引物。采用PCR从弓形虫RH株基因组DNA中分别扩增出编码GRA8的基因片段，克隆至pMD18-T载体；菌落PCR鉴定阳性克隆并测序分析；各组阳性克隆的质粒分别亚克隆至原核表达质粒pGEX-4T-2和真核表达载体pVAXl，分别转化大肠杆菌BL21和JM109,PCR和酶切鉴定转化菌落的插入序列；将构建的原核表达菌株经IPTG诱导，SDS—PAGE和免疫印迹分析融合蛋白的表达；将构建的真核重组表达质粒免疫小鼠，观察其诱导的抗体应答。结果 PCR扩增出GRA8基因的特异片段。各组阳性克隆的序列正确，并分别被亚克隆到原核表达质粒pGEX-4T-2和真核表达载体pVAXl上，构建了弓形虫致密颗粒抗原GRA8的原核和真核重组表达质粒；原核表达质粒在大肠杆菌中表达了GRA8的融合蛋白；真核重组表达质粒诱导小鼠产生了抗弓形虫抗原的抗体。结论以pGEX-4T-2和pVAX1为载体，分别成功构建了GRA8的原核和真核重组表达质粒     

弓形虫pc-DNA3-ROP2核酸疫苗的研制

目的 研究廉价有效的抗弓形虫核酸疫苗,以用于人类和畜类弓形虫病的防治。方法 收集、纯化RH株弓形虫速殖子,提取基因组DNA;根据ROP2基因序列,设计合成ROP2基因引物,应用PCR、酶切、连接、测序等技术,扩增ROP2基因片段,克隆于真核表达载体pc-DNA3质粒中,制备抗弓形虫pc-DNA3-ROP2核酸疫苗。应用该疫苗免疫小鼠,通过ELISA检测血清抗体,Western blot鉴定该疫苗的免疫原性;应用RH株弓形虫进行动物攻击实验,评价其安全性及免疫保护性。结果 以弓形虫基因组DNA为模板,PCR扩增出1.7 kb ROP2基因片段,克隆于pc-DNA3质粒中,成功构建了pc-DNA3-ROP2重组质粒。测序结果显示,重组质粒包含了ROP2蛋白基因读码框内的完整序列,能完整表达ROP2的抗原蛋白。应用该疫苗免疫小鼠,能诱导产生强烈的细胞、体液免疫反应,使实验组小鼠攻虫感染后的存活时间明显延长,攻虫感染192 h后的存活率为77.8%,空质粒及PBS对照组存活率为0,差异有统计学意义（P〈0.001）;实验全程免疫小鼠未发生毒性及异常反应。结论 该核酸疫苗具有很强的免疫原性,安全可靠,能诱导小鼠产生良好的免疫保护作用,具有很好的开发应用价值。     

截短弓形虫表面抗原SAG2C的基因克隆、蛋白表达及其初步应用

目的克隆截短的弓形虫表面抗原SAG2C基因,在大肠埃希菌中表达SAG2C蛋自,并探讨其在弓形虫病诊断中的应用。方法对已知的弓形虫SAG2C基因序列进行部分取舍,用RT-PCR技术从弓形虫Prugniaud（PRU）株的总RNA中扩增截短的SAG2基因片段,插入载体pET32a（＋）中,转化大肠埃希菌BL21,IPTG诱导表达,应用westemblot和EI—ISA检测重组表达蛋白的免疫反应性。用重组SAG2C蛋自ELISA祛检测弓形虫感染血清特异抗体．观察初步应用效果。结果从弓形虫PRU株总RNA中扩增出截短的SAG2CC基因片段,成功构建了重组表达质粒pET32a（＋）-tSAG2C;该重组质粒经IPTG诱导能表达可溶性大小为51ku的SAG2C蛋白。Western blot显求重组SAG2C能被弓形虫感染小鼠血清识别;以重组SAG2C蛋门、重组SAG1蛋白及BAG1蛋白ELISA检测精神病患者血清弓形虫抗体,阳性率分别为8．07%（23／285）、4．56％（13／285）和7．37％（21／285）,差异无统计学意义（P〉0．05）。结论成功构建了重组质粒pET32a（＋）-tSAG2C,表达的融合蛋白具有免疫反应性,具有用于弓形虫感染诊断的潜在价值。     

弓形虫SAG1单基因疫苗与SAG1-ROP2复合基因疫苗的免疫效果观察

目的构建弓形虫主要表面抗原SAG1单价基因疫苗及其与棒状体蛋白ROP2的复合基因疫苗,接种BALB/c小鼠,观察疫苗的免疫保护性。方法构建重组质粒pcDNA3.1SAG1及pcDNA3.1SAG1ROP2。将两核酸疫苗分别免疫小鼠,ELISA法检测血清IgG抗体、IFNγ、IL4;流式细胞仪测定T细胞亚群;弓形虫速殖子腹腔攻击感染观察小鼠生存时间。结果获得pcDNA3.1SAG1、pcDNA3.1SAG1ROP2重组质粒;pcDNA3.1SAG1ROP2组小鼠IgG抗体(P<0.05)、IFNγ(P<0.01)及CD8+细胞比例(P<0.05)均高于pcDNA3.1SAG1组;实验组组均未测到IL4;复合基因组感染弓形虫后生存时间较单基因组延长(P<0.01)。结论弓形虫不同生活阶段的抗原基因复合疫苗较单基因疫苗具有更好的免疫保护性。     

弓形虫表面抗原SAG4基因的克隆、表达和鉴定

目的 对弓形虫表面抗原SAG4基因进行克隆、表达和免疫反应性分析。 方法 根据弓形虫RH株SAG4基因序列（GenBank登录号为AF340224.1）设计引物,体外扩增目的基因,并将其克隆至pMD19-T载体,经PCR和双酶切鉴定并测序,亚克隆至pET28a（+）质粒,转化大肠埃希菌BL21,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析SAG4重组蛋白的表达情况,蛋白质印迹（Western blotting）分析该蛋白与弓形虫慢性感染小鼠血清的免疫反应性。 结果 扩增获得的目的基因片段为537 bp。生物信息学分析表明重组基因编码产物为弓形虫SAG4抗原,是一种外膜蛋白。重组菌经IPTG诱导后,以包涵体的形式稳定表达SAG4。Western blotting分析结果表明,诱导表达的蛋白能被弓形虫慢性感染小鼠血清识别,其相对分子质量（Mr）约为18 740。 结论 重组蛋白SAG4有一定的免疫反应性。     

弓形虫缓殖子期特异性抗原1基因的克隆、表达及对重组抗原的免疫反应性分析

目的 克隆与表达弓形虫缓殖子期特异性抗原1(BAG1)的基因,并分析重组抗原的免疫反应性。方法 诱导体外培养的弓形虫RH株速殖子向缓殖子转化,用RT-PCR法从缓殖子期弓形虫扩增BAG1基因片段,进行序列分析;构建表达重组质粒pET32a(+)-BAG1,转入大肠埃希菌BL21(DE3)中诱导表达;表达蛋白经次氨基三乙酸镍(Ni-NTA)琼脂糖亲和层析纯化后,蛋白质印迹(Western blotting)分析与ELISA分析其免疫反应性。 结果 从缓殖子期弓形虫克隆的BAG1基因长690 bp,构建的重组质粒pET32a(+)-BAG1经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,可高效表达重组BAG1。Western blotting分析显示纯化的重组BAG1(SAG1)能被弓形虫慢性感染血清识别。ELISA结果表明重组BAG1抗原检测350份人血清弓形虫IgG抗体的阳性率为17.4％,显著高于重组SAG1抗原的阳性率12.6％（P＜0.05）。 结论 原核表达的重组BAG1抗原具有特异的免疫反应性。     

弓形虫表面抗原SAG2基因的克隆表达纯化及鉴定

目的构建弓形虫表面抗原2(SAG2)基因重组质粒并在大肠埃希菌中表达。方法根据SAG2基因序列设计并合成引物,用PCR法从弓形虫基因组DNA中扩增SAG2基因片段,再克隆到p GEX-4T载体中,构建重组质粒。重组质粒经酶切鉴定并测序后,在大肠埃希菌BL21中诱导表达,产物经SDS-PAGE分析并纯化,以Western blotting分析其反应原性。结果 SAG2基因PCR产物大小约为561 bp,与预期相符。重组质粒经酶切及PCR鉴定构建成功,测序结果与已知序列吻合。重组质粒转化菌经IPTG诱导后表达的SAG2融合蛋白分子量约为47 ku,该蛋白可被GST标签抗体识别。结论成功重组了弓形虫SAG2基因,表达蛋白具有反应原性。     

弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4的构建

目的构建弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4。方法根据弓形虫RH标准株SAG1和SAG4基因序列分别设计一对特异引物(分别含有HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ内切酶位点),PCR扩增目的基因,并将这两段基因分别克隆至PEGM-T Easy载体,经菌落PCR和双酶切鉴定;用HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ分别双酶切PEGM-T Easy-SAG1、PEGM-T Easy-SAG4和pVAX1,得到含内切酶位点的SAG1和SAG4基因片段,分别与pVAX1连接,构建pVAX1-SAG1和pVAX1-SAG4真核表达质粒,经菌落PCR和双酶切鉴定。结果PCR扩增得到目的基因SAG1和SAG4,测序结果分别与弓形虫RH标准株SAG1和SAG4基因比较,符合率均为100%。构建PEGM-T Easy-SAG1、PEGM-T Easy-SAG4克隆质粒和pVAX1-SAG1、pVAX1-SAG4真核表达质粒,分别经菌落PCR和双酶切鉴定,显示722bp的SAG1基因片段和511bp的SAG4基因片段均插入载体PEGM-T Easy和pVAX1中。结论成功克隆了pVAX1-SAG1、pVAX1-SAG4真核表达质粒,为弓形虫基因疫苗应用研究奠定了基础。     

弓形虫抗原与霍乱毒素A2/B亚基复合基因在小鼠体内诱导的免疫反应

目的　观察弓形虫主要表面抗原SAG1、SAG2 与霍乱毒素A2 /B亚基复合基因真核质粒经肌肉免疫小鼠所诱导的免疫反应。方法　将SAG1基因、SAG2 基因及CTXA2 /B基因定向连接插入真核表达质粒pcDNA3.1,经酶切及测序,获得pcDNA3.1 SAG1 SAG2 及pcDNA3.1 SAG1 SAG2 CTXA2 /B的重组子;碱裂解法大量制备经肌肉注射免疫BALB/c鼠,每只鼠经后腿肌肉注射质粒10 0 μg ,每2周免疫1次,共3次,以PcDNA3.1空质粒注射组及PBS组为对照,分别于每次免疫前断尾取血和免疫后4周取小鼠脾脏测定T淋巴细胞增殖活性及NK细胞活性,ELISA法测定IgG抗体。结果　免疫组小鼠的IgG抗体水平明显提高,NK细胞杀伤活性和T细胞增殖活性也明显增强。免疫鼠抗攻击感染的时间延长。结论　含有霍乱毒素的复合基因免疫小鼠后体液免疫和细胞免疫水平均有提高。     

Biological role of surface Toxoplasma gondii antigen in development of vaccine

AIM: To analyze the biological role of the surface antigen of Toxoplasma gondii (T gondii) in development of vaccine. METHODS: The surface antigen of T gondii (SAG1) was expressed in vitro. The immune response of the host to the antigen was investigated by detection of specific antibody reaction to SAG1 and production of cytokines. Mice were immunized with recombinant SAG1 and challenged with lethal strain of T gondii RH. The monoclonal antibody to r-SAG1 was prepared and used to study the effects of SAG1 on T gondii tachyzoites under electromicroscope. RESULTS: The mice immunized with recombinant SAG1 delayed death for 60 h compared to the control group. The recombinant SAG1 induced specific high titer of IgG and IgM antibodies as well as IFN-γ, IL-2 and IL-4 cytokines in mice. In contrast, IL-12, IL-6 and TNF-αwere undetectable. When T gondii tachyzoites were treated with the monoclonal antibody to r-SAG1, the parasites were gathered together, destroyed, deformed, swollen, and holes and gaps formed on the surface. CONCLUSION: SAG1 may be an excellent vaccine candidate against T gondii. The immune protection induced by SAG1 against T gondii may be regulated by both hormone- and cell-mediated immune response.     

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a > IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.     

刚地弓形虫ROP2-P30复合重组蛋白疫苗对BALB／c小鼠免疫保护性的研究

目的用刚地弓形虫ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠观察对机体的影响,为研制安全有效的弓形虫疫苗奠定基础。方法PCR方法从刚地弓形虫基因组中扩增出ROP2和P30片段,将这两个片段分别亚克隆至pET-30a原核表达载体中,表达出ROP2-P30复合重组蛋白。用蛋白疫苗免疫BALB/c小鼠,ELISA检测免疫小鼠后体液中抗体和细胞因子的变化,观察攻击试验小鼠的的死亡率。结果ROP2-P30复合重组蛋白疫苗免疫BALB/c小鼠诱导机体产生大量的IgM、IgG抗体和IFN-γ、IL-2及IL-4细胞因子。攻击试验表明,复合重组蛋白免疫组和对照组相比,小鼠的存活时间明显延长。结论ROP2-P30复合重组蛋白免疫BALB/c小鼠诱导其产生高水平的体液和细胞免疫应答,该复合重组蛋白是抗刚地弓形虫感染的候选疫苗之一。     

Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents

Immunization with DNA can induce humoral and cell-mediated immune responses, both of which are important in conferring immunity to Toxoplasma gondii. The efficacy of genetic vaccination with a cDNA encoding the T. gondii SAG1 (P30) surface antigen was evaluated. Sera of immunized mice showed recognition of T. gondii tachyzoites by immunofluorescence and exhibited high titers of antibody to SAG1 by ELISA. SAG1-stimulated splenocytes from vaccinated mice produced primarily interferon-gamma and interleukin-2. Vaccinated mice survived challenge with 80 tissue cysts of ME49 strain, whereas all control mice died; challenge with 20 tissue cysts resulted in fewer brain cysts, compared with controls. Challenge of vaccinated rats with VEG strain oocysts resulted in a reduction in brain cysts. No protection was observed when mice were challenged with the highly virulent RH strain tachyzoites. These results suggest that nucleic acid vaccination can provide protection against T. gondii infection in mice.