IL-18增强日本血吸虫DNA疫苗Sj23的免疫保护效果

将小鼠IL-18基因和日本血吸虫Sj23膜蛋白基因分别插入pVAX1载体,构建真核表达质粒pVAX/mIL-18和pVAX/Sj23,联合或单独免疫小鼠,以pVAX1空载体作对照,免疫2次,间隔2周,2次免疫后4周攻击日本血吸虫尾蚴.体液和细胞免疫检测结果表明,联合免疫组能诱导小鼠产生较强的抗日本血吸虫成虫可溶性抗原(SWAP)IgG,较高水平的IFN-γ和IL-2.攻虫试验表明,联合免疫小鼠成虫减虫率和肝脏减卵率分别达41.6%和49.4%,明显高于pVAX/Sj23单独免疫组(减虫率和肝脏减卵率分别为26.5%和41.4%).以上试验结果表明,IL-18能明显增强Sj23 DNA疫苗在小鼠体内的免疫应答,并且产生较强的保护作用.     

日本血吸虫还原性辅酶I(SjNADH)基因的克隆表达及其免疫保护效果分析

为了评估日本血吸虫还原性辅酶I(SjNADH)在小鼠体内诱导的免疫保护效果,应用PCR获得SjNADH基因片段,其开放阅读框为474 bp,编码157个氨基酸,并在大肠杆菌中成功表达,纯化得到该重组蛋白。Western blot结果显示,SjNADH在日本血吸虫童虫和成虫中的表达量稳定,SjNADH融合蛋白也能被免疫血清识别,具有较好的免疫原性。应用重组蛋白免疫小鼠能诱导产生较高的特异性抗体水平,并诱导了20.13%的减虫率和23.06%的肝脏减卵率。结果表明,获得的SjNADH重组蛋白在小鼠体内诱导产生了部分免疫保护,有作为血吸虫疫苗候选抗原分子的潜力。     

日本血吸虫重组IAP蛋白的免疫保护效果评估

为研究日本血吸虫凋亡蛋白抑制因子（inhibitor of apoptosis protein of Schistosoma japonicum,SjIAP）重组蛋白诱导BALB/c小鼠的免疫保护效果,利用PCR技术扩增SjIAP基因,构建重组表达质粒pET-28a（＋）-SjIAP,诱导表达重组SjIAP蛋白,并利用重组蛋白制备兔源多克隆抗体血清。然后,选用SjIAP重组蛋白免疫BALB/c小鼠,利用ELISA检测免疫小鼠血清中的特异性抗体水平,以及免疫小鼠脾脏淋巴细胞在SjIAP重组蛋白刺激后产生的细胞因子水平。强化免疫后,将小鼠进行血吸虫尾蚴攻虫试验,感染38 d,进行剖杀,计算虫体减虫率及肝脏减卵率。Western blot结果表明本研究制备的兔源多抗血清能特异性识别SjIAP重组蛋白。ELISA检测表明免疫SjIAP重组蛋白可诱导较高水平的IgG及IgG亚型（IgG1、IgG2a、IgG2b、IgG3）抗体和IFN-γ、IL-2及IL-4细胞因子。动物试验表明,免疫SjIAP重组蛋白的小鼠与PBS组相比分别获得了31.5%的减虫率和37.2%的肝脏减卵率。免疫SjIAP重组蛋白能诱导小鼠获得一定减虫和减卵保护效果,提示血吸虫凋亡蛋白抑制因子可作为抗血吸虫病的疫苗候选分子。     

日本血吸虫GSK3蛋白编码cDNA的克隆、表达及其重组蛋白的免疫保护研究

本研究利用生物信息学分析了日本血吸虫(Schistosoma japonicum,sj)Glycogen synthase kinase 3(GSK3)蛋白,并对其中一个GSK3蛋白的编码cDNA进行了克隆和原核表达,制备了特异性的多克隆抗体.同时,还初步评估了重组蛋白的免疫保护效果.生物信息学分析表明在日本血吸虫数据库存在两种GSK3蛋白,且其中一个SjGSK3在日本血吸虫不同发育时期均有转录.Western blot结果表明本研究制备的抗体能特异性识别日本血吸虫SjGSK3重组蛋白,表明该重组蛋白具有良好的免疫原性.动物实验表明免疫SjGSK3重组蛋白的动物与佐剂对照组比较分别获得了平均10.6％减虫率和40.5％肝脏减卵率.     

牦牛日本血吸虫Sjp38MAPK基因克隆、原核表达及免疫保护效果研究

为了分析牦牛日本血吸虫Sjp38MAPK重组蛋白的免疫效果,试验采用RT-PCR方法克隆出牦牛日本血吸虫Sjp38MAPK基因,构建重组表达载体pET-30a(+)-Sjp38MAPK,转化到BL21(DE3)中,IPTG诱导表达,采用SDS-PAGE和Western-blot方法检测其蛋白的表达情况。结果表明,获得约为55 ku的重组蛋白rSjp38MAPK,表达的重组蛋白Sjp38MAPK能被感染日本血吸虫的阳性血清识别,具有免疫原性。用纯化的重组蛋白免疫BALB/c小鼠后,以临床分离的日本血吸虫尾蚴腹部贴片攻毒,发现重组蛋白免疫组能降低小鼠的减虫率和肝脏减卵率,可诱导产生特异IgG抗体,具有较好免疫保护效果。这为进一步研究Sjp38MAPK蛋白功能及研制基因疫苗奠定基础。     

日本血吸虫多价核酸疫苗的构建及免疫保护试验

利用PCR技术获得日本血吸虫GST抗原基因,通过分子克隆技术将GST、FABP基因克隆至pVAX1载体,并将其克隆至舍有Sj23基因表达核的plRESneo栽体,构建重组质拉pVAX-GST、pVAX-GST-FABP、pIRESneo-Sj23和pIRESneo-GST-FABP-Sj23.将50只昆明小鼠随机分成5组,每组10只,分别以肌肉注射质粒pIRESneo-GST-FABP-Sjz3、plRESneo-Sj23、pVAX-GST、pVAX-GST-FABP和空白质粒plRESneo,50μg/只,免疫2次,每次间隔21 d.测定小鼠免疫前后血清抗体水平,30 d经腹部皮肤感染血吸虫尾蚴,45 d后剖杀小鼠,计数各组小鼠虫卵数及成虫数.结果表明.重组质粒pVAX-GST、plRESneo-Sj23、pVAX-GST-FABP和pIRESneo-GST-FABP-Sj23均能诱导小鼠产生特异性的IgG抗体,plRESnecrGST-FABP-Sj23免疫组所诱导的IgG水平最高;减虫率分别为26.1%、30.8%、33.2%和47.5%,减卵率分别为25.4%、45.0%、48.4%和69.8%.pIRESneo-GST-FABP-Sj23的减卵率和减虫率明显高于其他DNA疫苗.结果表明,日本血吸虫多价核酸疫苗能产生较强的免疫反应,并能获得较好的免疫保护效果.     

日本血吸虫基因重组抗原rSj06868对小鼠的免疫保护效果

为克隆、表达日本血吸虫假想蛋白SJCHGC068068编码基因（暂命名为Sj06868）并观察重组抗原的免疫预防效果。应用PCR技术从日本血吸虫7 d童虫、14 d童虫mRNA反转录制备的cDNA中克隆到Sj06868基因的46-438 bp DNA片段,BLASTn分析未发现其他物种中的同源性序列,也未发现其保守序列和相关功能结构域。以pET32a为表达载体、Sj06868基因在大肠杆菌Rosetta（DE3）中获得高效表达,表达产物rSj068068分子量为36 kDa,能被感染血吸虫14 d兔血清识别。将纯化的重组抗原与佐剂ESSAI ISA 206 CELL混合后免疫BALB/c小鼠,与佐剂对照组和PBS对照组相比,分别获得了31.63%、29.19%减虫率以及55.63%、56.27%肝脏虫卵减少率。用ELISA检测各组血清中抗rSj068068特异性抗体结果显示,免疫组在攻击感染前、佐剂对照组和PBS对照组在感染后均产生了较高水平的特异性抗体。本研究结果说明,Sj068068是日本血吸虫特有的童虫期高表达基因,rSj068068在抗血吸虫疫苗和血吸虫感染的诊断方面均有潜在应用价值。     

日本血吸虫编码抱雌沟蛋白保守区cDNA片段的克隆表达及其免疫保护功能

本研究根据曼血吸虫抱雌沟蛋白（Gyncophoral canal protein)基因SmGCP保安区片设计－对引物,以日本血吸虫中国大陆株成虫mRNA为模板,用TR－PCR法扩增出一大小为868bp的基因片段。测序后分析序列推断该片段与SgGCP相应片段碱基同源性86．6％。说明所克隆的基因为编码日本血吸虫中国大陆株抱雌沟蛋白基因,推断的氨基酸序列同源性为84．1％,将其克隆到表达载体pET28（a)中,在大肠杆菌BL21中获得了较高量的融合表达,表达产物分子量为29KD。利用因吸虫成虫粗抗原免疫大白兔所得抗血清对该表表达产物进行Westernblto检测,在预测位置出现了明显的识别条带,显示了该表达产物良好的抗原性。将该表达产物用佐剂处理免疫昆明系小鼠攻击血吸虫尾蚴,7周后剖杀小鼠冲虫,进行成虫和虫卵计数,其数虫率为35．2％,减卵率为37．8％,与对照组比较差异显著,初步证明血吸虫抱雌沟蛋白具有一定的免疫保护功能。     

日本血吸虫SjCHGC06822蛋白诱导BALB/c小鼠免疫保护效果观察

利用PCR技术扩增日本血吸虫Sj CHGC06822 ORF全长序列,以p ET28a(+)为载体构建重组质粒,大肠杆菌表达系统进行表达,His-Ni柱层析纯化r Sj CHGC06822/His蛋白后,生物信息学分析其可能结构,Western blot分析其抗原性。利用重组蛋白免疫BALB/c小鼠评估其免疫效果,ELISA分析血清抗体亚型变化。结果显示,获得日本血吸虫Sj CHGC06822的完整ORF序列591 bp,编码192个氨基酸,序列aa37~aa72含一个EF-hand手性域结构,无信号肽及跨膜结构。获得的Sj CHGC06822/His重组蛋白具有较好的抗原性,在2次独立BALB/c小鼠实验中,与PBS对照组相比,Sj CHGC06822/His蛋白免疫组诱导小鼠获得30.36%、40.26%减虫率和30.14%、12.28%肝脏减卵率,血清Ig G抗体免疫及感染后持续增高,Ig G2a和Ig G1水平随免疫次数增高,感染后出现下降,但Ig G2a/Ig G1(1)水平持续增加。本研究成功克隆了Sj CHGC06822基因,并成功表达纯化了r Sj CHGC06822/His蛋白,且免疫原性和反应原性良好。Sj CHGC06822蛋白免疫可以诱导BALB/c小鼠产生一定的保护力,诱导宿主的免疫应答更偏向于Th1型。     

pVAX1/SjTSP2 DNA疫苗免疫预防小鼠日本血吸虫病的效果评估

为评估SjTSP2分子作为日本血吸虫DNA疫苗候选抗原分子的潜力,本实验扩增了编码该分子的DNA片段,构建了重组真核质粒pVAX1/SjTSP2,检测其在哺乳动物细胞中表达情况后,通过基因枪轰击法免疫小鼠,应用ELISA、流式细胞术等检测重组质粒诱导的免疫反应,计算减虫减卵率,评估对小鼠日本血吸虫病的免疫保护效果。间接免疫荧光试验结果显示该质粒能在293T细胞中表达。质粒免疫小鼠诱导了显著升高的特异性IgG抗体和IFN-γ、IL-4等细胞因子。小鼠免疫保护试验结果表明,免疫组小鼠虫荷数较PBS对照组增加12.4%(P=0.368),但肝组织虫卵减少10.01%(P=0.486)。说明制备的DNA疫苗能刺激小鼠产生较强的免疫反应,但诱导的免疫保护作用并不理想,为血吸虫DNA疫苗研究提供了参考数据。     

Persistence of the protective immunity to Schistosoma japonicum in Chinese yellow cattle induced by recombinant 26kDa glutathione-S-transferase (reSjc26GST)

To observe the long lasting effect of the recombinant Sj26GST sub-unit vaccine against Schistosoma japonicum in cattle, animals aged from 5 to 12 months were vaccinated with reSjc26GST, and were challenged by natural infection 6 months or 12 months after vaccination. Worm burdens per cattle and egg burden in tissue (per gram) of cattle with or without vaccination were compared. The results showed that anti-reSjc26GST antibodies were produced in vaccinated cattle. Following natural infection, the vaccinated and the control non-vaccinated cattle were all found to be infected with S. japonicum. A 30% reduction in worm number was observed in the vaccinated cattle when compared with the control cattle. The anti-fecundity effect was characterized by an average of 60% decrease in eggs deposited in the liver of vaccinated cattle; such a decrease is obviously very significant. In addition to the anti-fecundity effect induced in the vaccinated cattle, the number of miracidum hatched per 50 g faeces and the number of eggs released in intestinal tissues per gram were reduced or decreased. Results suggested that the immune responses induced by reSjc26GST in cattle were similar to that in buffaloes and in pigs. In addition, our result demonstrated that the lasting effect of immunity to S. japonicum induced in cattle after vaccination with reSjc 26 GST could persist at least 12 months.     

Peroral infection of pigs with Schistosoma japonicum cercariae.

Infections with the zoonotic trematode, Schistosoma japonicum in pigs serves as a valuable model for studying natural definitive host/parasite relationships and a model for human schistosomosis japonica. In the present study the efficiency of a peroral infection route was compared with that of an intramuscular route of infection. Eleven specific pathogen-free Danish Landrace/Yorkshire/Duroc crossbred male and female pigs were divided into two groups of five and six pigs, respectively. Each pig was given 1000 cercariae, either placed in droplets on the mucosa in the buccal cavity, or as medium-suspended cercariae injected into musculus biceps femoris of one of the hindlegs. Ten weeks post infection, all pigs were killed with pentobarbital and the venous system perfused. Worm burdens and liver egg counts were determined and worm fecundity was calculated. S. japonicum infections were established in all individuals in both groups of pigs. When comparing the two groups, the peroral group had significantly higher number of immature worms, whereas the intramuscularly infected group had significantly more worm nodules. However, no difference was seen in total number of worms. No statistical significant differences were found in neither tissue egg counts nor worm fecundity when comparing the two groups. The results from the present study showed a delay in maturation of infection following a peroral infection as compared with an intramuscular infection, but comparability was seen between overall worm establishment and egg production.     

Cytokine mRNA profiles in pigs exposed prenatally and postnatally to Schistosoma japonicum

The pig is a natural host for Schistosoma japonicum and a useful animal model of human infection. The aim of the present study was to assess the differences between the cytokine profiles in prenatally or postnatally S. japonicum exposed pigs. Seven prenatally exposed pigs, 7 postnatally exposed pigs and 4 uninfected control pigs were compared 27 weeks post exposure. Variables included worm burdens, tissue egg counts, liver pathology and mRNA levels of IL-2, IL-4, IL-10, IL-12, TNF-alpha, TGF-beta1 and IFN-gamma in the liver and the caecum, assessed by RT-PCR. Infection intensity and level of septal fibrosis were significantly higher in the postnatal group compared to the prenatal group (P < 0.05). A significant increase of IL-4 (P < 0.01), IL-10 (P < 0.01), IL-12 (P < 0.01) and TNF-alpha (P < 0.05) mRNA level was also observed in the caecum of prenatally infected animals compared to the control group (P < 0.01). The prenatal group showed higher levels of TGF-beta1 in the liver compared with the postnatally infected group (P< 0.05) and the control group (P< 0.01). This suppressive immune response correlated with previously reported low hepatic pathogenesis in prenatally exposed pigs.     

Evaluation of coproantigen diagnosis for natural Echinococcus multilocularis infection in red foxes.

The validity of a coproantigen ELISA for Echinococcus multilocularis was evaluated by comparison of three diagnostic methods; autopsy, egg examination and the ELISA. Of 71 foxes, 39 were found to be infected with the cestode at autopsy. The overall mean of worm burdens was 3,451, but the number varied (1-34,522). The ELISA could detect 94.9% (37/39) of the worm positives and there were no false-positives. Two false-negatives were infected with 1 and 4 cestodes, whereas 3 cases with similar worm burdens (2, 4 and 6 worms) were diagnosed as positives. This indicates the detection limit of the assay may be equivalent to less than 10 (in the worm burden). On the other hand, egg examination showed low sensitivity (43.6%, 17/39). These results suggest the ELISA has a potential to replace for the conventional methods.     

日本血吸虫谷胱甘肽S－转移酶小鼠免疫试验

用谷胱甘肽琼脂糖亲和层析柱纯化中国大陆株日本血吸虫成虫谷胱甘肽Ｓ－转移酶（ＧＳＴ）。把纯化的ＧＳＴ结合福氏性剂免疫了二批达ＢＡＬＢ／ｃ小鼠，获得了２９．５８～３２．７１％的减虫率和５２．９４～６８．１３％的粪便减卵率。ＥＬＩＳＡ试验表明免疫小鼠产生了体液免疫应答，免疫印迹试验（ＷｅｓｔｅｒｎＢｌｏｔｔｉｎｇ）显示日本血吸虫水溶性成虫抗原和ＧＳＴ抗原的２６、２８ｋＤ蛋白被免疫小鼠血清所识别。     

吡喹酮注射剂早期治疗小鼠血吸虫病效果的初步研究

为探索吡喹酮新制剂早期治疗血吸虫病效果,对人工感染日本血吸虫20d的小鼠皮下给药进行减虫率、肝脏减卵率统计,并在不同时间点对口服组和皮下注射吡喹酮新制剂组的未感染小鼠在给药后采集血浆,高效液相色谱法建立药时曲线。结果显示新制剂600mg/kg剂量治疗的3次试验的减虫率分别为72.88%、82.69%和56.7%,肝脏减卵率分别为81.7%、94.8%和70.7%,对感染20d期别的血吸虫有较好效果。而高效液相色谱法检测血药浓度显示,新制剂经皮下给药方式可使有效杀虫浓度(约0.3μg/mL)持续10d以上,具有缓释作用。吡喹酮新注射制剂能杀灭20d童虫,减少血吸虫病的危害,对血吸虫病的防治有较大的意义。