Congenital infection of rabbits with Schistosoma japonicum and protective immunity of offspring

Background Recently congenital infection with Schistosoma japonicum (S. japonicum) has been domonstrated in pigs, rabbits, mice and dogs. We explored the rabbit as an animal model for the congenital infection of schistosomiasis japonica and assessed the effect of a congenital S. japonicum infection on the resistance of rabbit kittens to a postnatal challenge infection.Methods Sixteen pregnant New Zealand white rabbits were infected with a single dose of S. japonicum cercariae. The exposed animals were divided into three groups according to the gestation age at the time of infection. Diagnosis of prenatally acquired S. japonicum infection in the rabbit kittens was primarily based on serological tests in combination with parasitological and histopathological findings. Congenitally infected kittens were challenged percutaneously with 100 S. japonicum cercariae to assess the effect of a congenital S. japonicum infection on kitten resistance to a postnatal challenge infection.Results The overall prevalence of congenital infection in offspring of infected mothers was 20% (12/60). The congenital infection rate in group L (late gestation) was much higher than in group E (early gestation) and group M (mid-gestation) (P<0.05). After a postnatal challenge infection, prenatally infected kittens had a 54.66% worm reduction rate, 41.45% egg reduction rate, and 51.76% granuloma size reduction rate compared to nave kittens.Conclusions This study demonstrates the possibility of congenital infection of S. japonicum in rabbits and the resistance of congenitally infected kittens to a postnatal challenge infection. These results have important implications not only for epidemiological investigations, but also in designing government control programs for schistosomiasis.     

重组日本血吸虫SjGT—Sj32蛋白诱导小鼠保护免疫的研究

应用基因工程技术分别表达出ＳｊＧＳＴ－Ｓｊ３２,Ｓｊ３２和ＳｊＧＳＴ,用于保护性免疫的动物实验及其体液免疫机制的初步探讨。结果：与对照组相比,重组ＳｊＧＳＴ－Ｓｊ３２组,Ｓｊ３２组,Ｓｊ３２＋ＳｊＧＳＴ组均有明显的减虫效果。上述三组及ＳｊＧＳＴ组的肝脏虫卵计数的均较对照组为少。提示重组Ｓｊ３２ｋＤ蛋白可诱导小鼠产生明显的抗日本血吸虫攻击感染的保护性免疫及抗生殖免疫效果,而ＳｊＧＳＴ仅表现出一定的抗     

日本血吸虫尾蚴抗原模拟表位的筛选及其免疫保护性

目的 筛选日本血吸虫尾蚴抗原的模拟表位，探讨其对日本血吸虫的免疫保护性。方法 用粗提日本血吸虫尾蚴抗原的抗体IgC作配体筛选噬菌体12肽库，按“吸附－洗脱－扩增”的过程进行三轮筛选；随机挑取单个噬菌体克隆进行Dot-ELISA检测；用混和噬菌体克隆免疫小鼠3次，攻击感染45d后剖杀冲虫，计算虫数及肝卵数。结果 经三轮筛选，特异性噬菌体得到富集，挑取11个噬菌体克隆经Dot-ELISA鉴定，均与日本血吸虫尾蚴抗原免疫血清呈特异性反应。与对照组相比，混和噬体克隆免疫小鼠的减虫率为18.79%，减卵率为38.00%。结论 利用噬菌体随机肽库技术获得了日本血吸虫尾蚴抗原的模拟表位，这些表位能诱导对日本血吸虫的保护性免疫。     

日本血吸虫Rho GTPase DNA疫苗及亚单位疫苗的构建和分析

目的 构建日本血吸虫大陆株Sj-Rho GTPase-like真核及原核表达重组质粒，并进行鉴定分析。方法 用PCR法将Sj-Rho GTPase-like基因从已剪切阳性克隆中扩增出来，亚克隆至pcDNA3.1和pGEX-5X-3中，分别经PCR、双酶切、测序、SDS-PAGE和Western blot等方法鉴定。结果 PCR和测序均证明Sj-Rho GTPase-like基因疫苗构建成功，重组蛋白经SDS-PAGE电泳可观察到与预期分子量相应的条带，转印后可被水牛感染血清识别。结论 成功构建了Sj-Rho GTPase-like基因的两种重组载体，为保护性免疫研究打下基础。     

重组日本血吸虫SjGST-Sj32蛋白诱导小鼠保护性免疫的研究

应用基因工程技术分别表达出ＳｊＧＳＴＳｊ３２,Ｓｊ３２和ＳｊＧＳＴ,用于保护性免疫的动物实验及其体液免疫机制的初步探讨。结果：与对照组相比,重组ＳｊＧＳＴＳｊ３２组,Ｓｊ３２组,Ｓｊ３２＋ＳｊＧＳＴ组均有明显的减虫效果（Ｐ＜０．０５）。上述三组及ＳｊＧＳＴ组的肝脏虫卵计数均较对照组为少（Ｐ＜０．０１）。提示重组Ｓｊ３２ｋＤ蛋白可诱导小鼠产生明显的抗日本血吸虫攻击感染的保护性免疫及抗生殖免疫效果,而ＳｊＧＳＴ仅表现出一定的抗生殖免疫效果。     

一种新型日本血吸虫疫苗(童虫活细胞)诱导小鼠产生有效保护性免疫力的研究

目的观察日本血吸虫童虫活细胞诱导小鼠产生的保护性免疫效果.方法采用日本血吸虫肝期童虫的活细胞和虫体组织碎片,在未加佐剂条件下分别接种昆明小鼠,每组10只,每2周接种1次,共4次,末次接种后第7天攻击感染血吸虫尾蚴30条/鼠,感染后第42天解剖小鼠,与对照组比较检测和分析虫体发育数、虫体大小、鼠肝病变及其组织内虫卵肉芽肿大小、血清中特异性抗体水平和IgG2a/IgG1亚类比值.结果细胞接种组和虫体碎片接种组分别与PBS注射组比,虫体发育数分别下降67.6%%和46.3%;肝组织中虫卵数(LEPG)在细胞接种组和虫体碎片接种组中分别减少95.4%和64.8%;在细胞接种鼠组肝表面卵结节和虫卵肉芽肿及雄虫长度均显著小于其他两组者;特异性IgG2a/IgG1比值≥2,但总抗体水平低于虫体接种组.结论该研究首次建立了一种血吸虫细胞型疫苗研究模型,初步结果表明,在不加佐剂条件用血吸虫童虫活细胞可诱导小鼠产生显著的抗攻击感染免疫力,其机制可能主要为Th1介导的细胞免疫应答.     

Investigation of recombinant Schistosoma japonicum paramyosin fragments for immunogenicity and vaccine efficacy in mice

Schistosoma japonicum paramyosin, a 97 kDa myofibrillar protein, is a recognized vaccine candidate against schistosomiasis. To improve its expression and to identify protective epitopic regions on paramyosin, the published Chinese Schistosoma japonicum paramyosin cDNA sequence was redesigned using Pichia codon usage and divided into four overlapping fragments （fragments 1, 2, 3, 4） of 747, 651, 669 and 678 bp, respectively. These gene fragments were synthesized and expressed in Pichia pastoris （fragments 2 and 3） or E. coli （fragments 1 and 4）. The recombinant proteins were produced at high level and purified using a two-step process involving Ni-NTA affinity chromatography and gel filtration. BALB/c mice were immunized subcutaneously three times at 2-week-intervals with the purified proteins formulated in adjuvant Quil A. The protein fragments were highly immunogenic, inducing high, though variable, ELISA antibody titres, and each was shown to resemble native paramyosin in terms of its recognition by the anti-fragment antibodies in Western blotting. The immunized mice were subjected to cercarial challenge 2 weeks after the final injection and promising protective efficacy in terms of significant reductions in worm burdens, worm-pair numbers and liver eggs in the vaccinated mice resulted. There was no apparent correlation between the antibody titres generated and protective efficacy, as all fragments produced effective but similar levels of protection.     

日本血吸虫抗原的表位模拟肽筛选及免疫保护性

目的　筛选日本血吸虫雄虫抗原的表位模拟肽 ,并探讨其诱导小鼠抗日本血吸虫的免疫保护性。　方法　用日本血吸虫可溶性雄虫抗原的IgG对噬菌体随机 1 2肽库进行亲和筛选。 3轮筛选后 ,经Dot-ELISA检测获得阳性噬菌体克隆 ,并用混合特异性噬菌体克隆免疫小鼠及进行抗日本血吸虫免疫保护性分析。　结果　 1 8个特异性噬菌体克隆均显示有抗原性 ,其噬菌体混合克隆免疫诱导小鼠产生了特异性抗体 ,以及 31 72 %的减虫率和 51 54%的减卵率 ,与对照组比较差异有显著性 (P <0 0 0 1 )。　结论　筛选噬菌体肽库获得的日本血吸虫可溶性雄虫抗原的表位模拟肽分子能诱导小鼠产生抗日本血吸虫的保护性免疫。     

日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3的构建及其保护性免疫研究

目的构建日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3,用以免疫小鼠,观察其在小鼠抗血吸虫感染中的免疫保护作用。方法根据质粒pGEX-4T-1中SjGST-ORF和SjFABP基因序列,利用基因重组、PCR等技术将SjGST和SjFABP编码基因拼接在一起,得到融合基因SjGST-FABP,将融合基因SjGST-FABP定向克隆到pcDNA3多克隆位点上,转化大肠杆菌,经质粒扩增和DNA序列测定后,进行小鼠动物免疫和日本血吸虫尾蚴攻击感染及免疫保护性评价。结果成功构建了日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3。免疫小鼠获得42.39%的减虫率和56.09%肝减卵率（P〈0.05）。结论日本血吸虫DNA多价疫苗SjGST-FABP/pcDNA3可诱导部分抗血吸虫尾蚴攻击感染的免疫保护效果,具有疫苗研究与开发价值。     

日本血吸虫SjC23-Hsp70 DNA 疫苗与IL-12对水牛保护性作用的研究

目的:研究日本血吸虫中国大陆株23 kD 膜蛋白-热休克蛋白(SjC23-Hsp70)DNA 疫苗联合佐剂白细胞介素12(IL-12) 质粒DNA 对水牛的免疫保护作用。方法:将血吸虫病非流行区8~10 月龄健康水牛45 头随机分为A组(SjC23-Hsp70+IL-12)、B 组(SjC23+IL-12) 和C 组(pVAX+IL-12),每组15 头。每头牛经肩部肌注免疫3 次,每次间隔28 d。末次免疫后28 d,每头牛感染日本血吸虫尾蚴1000 条。解剖前2 天及当天分别收集粪便1 次,用定量法检测虫卵和毛蚴数。攻击感染后56 天解剖所有水牛,经胸主动脉灌冲法收集成虫,计数成虫数,检测每克肝组织虫卵数。结果:A,B 组与C 组相比,分别获得45.70%和26.61%的减雌率,44.51%和25.84%的减虫率,41.10%和31.63%的减粪卵率,48.11%和38.07%的减毛蚴率及43.39%和31.95%的减肝卵率。A 组的5 个率均比B 组高(P<0.05)。结论:用SjC23-Hsp70 DNA 疫苗和IL-12 联合免疫水牛可获得明显的免疫保护作用。     

Sj31与鼠IFN-γ基因融合DNA疫苗免疫保护效果的研究

目的构建表达mIFN-γ与Sj31抗原融合蛋白的DNA疫苗,并探讨其免疫保护作用。方法在Sj31基因上游引物与mIFN-γ下游引物的5’分别设计与克隆载体相匹配的酶切位点,在mIFN-γ上游引物与Sj31基因下游引物的5’设计相同的酶切位点。分别进行PCR扩增,再通过引物的5’端相同的酶切位点进行2个PCR产物的酶切与连接,以连接产物为模板,应用Sj31的上游引物和mIFN-γ下游引物进行PCR扩增,将此次PCR产物经常规方法克隆入载体pcDNA3.0(简称为pc),构建成多价DNA疫苗pc-Sj31-mIFN-γ。对重组质粒鉴定后,大量制备纯化质粒免疫昆明小鼠,48只小鼠分为A、B、C、D4组,对照组的小鼠于股四头肌注射质粒pcDNA3.0100μg,3个免疫组的小鼠分别注射pc-Sj31、pc-mIFN-γ和pc-Sj31-mIFN-γ质粒DNA各100μg。在0、2、6周免疫共3次,第3次免疫后2周,每只小鼠经腹部贴片感染尾蚴40±1条,感染后第42天剖杀,计数各小鼠检获成虫数及肝卵数。结果pc-Sj31-mIFN-γ(D组)免疫组的减虫率和肝组织减卵率分别为28.56%和60.20%,但是表达鼠mIFN-γ与Sj31抗原融合蛋白的DNA疫苗并未明显优于单纯表达日本血吸虫组织蛋白酶B(Sj31)DNA疫苗的免疫保护作用。结论pc-Sj31-mIFN-γ多价DNA疫苗构建成功,但其诱导小鼠抗血吸虫病免疫保护效果不明显优于pc-Sj31。     

日本血吸虫Sj31核酸疫苗联合IFN-γ重组质粒诱导小鼠保护性免疫的研究

目的　探讨干扰素 γ重组质粒对日本血吸虫组织蛋白酶B核酸疫苗在小鼠抗血吸虫作用的影响。　方法　将小鼠干扰素 γ基因PCR扩增片段克隆入真核表达载体pCDNA3 1以构建重组真核表达质粒pCDNA3 1 IFN γ ,并与日本血吸虫组织蛋白酶B真核表达质粒VR10 12 Sj3 1一同免疫小鼠。小鼠分为 4组 ,其中实验组每鼠同时肌注VR10 12 Sj3 1及pCDNA3 1 IFN γ各 10 0 μg ,3个对照组分别为VR10 12 Sj3 1肌注 10 0 μg ,pCDNA3 1 IFN γ肌注 10 0 μg和载体VR10 12及pCHAN3 1肌注各 10 0 μg。共免疫 3次 ,每次间隔 2周。于末次免疫后两周免疫组化检测表达质粒在小鼠肌细胞的表达 ,于末次免疫后 3周经小鼠皮肤攻击感染 40± 1条日本血吸虫尾蚴。 45d后杀小鼠计算减虫率。　结果　VR10 12 Sj3 1及pCDNA3 1 IFN γ均在小鼠肌细胞表达 ,日本血吸虫Sj3 1核酸疫苗联合IFN γ重组质粒免疫可诱导小鼠产生 2 7 3 7%的减虫率 ,与日本血吸虫Sj3 1核酸疫苗单独免疫组比较减虫率显著 (P <0 0 5 )。　结论　IFN γ表达质粒能增强日本血吸虫组织蛋白酸B核酸疫苗的抗日本血吸虫的作用     

香菇多糖对日本血吸虫DNA疫苗pVIVO2-Sj14-Sj23的增效作用

血吸虫病是一个严重的公共卫生问题,血吸虫疫苗的研究已有半个多世纪,其中核酸疫苗,包括混合或多价DNA疫苗。但疫苗候选分子诱导的保护力较弱,很少超过50％减虫率。为此,要继续寻找新的疫苗抗原分子,同时应重视筛选可用于人体的疫苗佐剂,进一步提高其保护力。多年来,人们不断地探索希望能找出安全有效的佐剂,可是实际上并不如意。     

钩端螺旋体DNA疫苗pcD-flaB的构建及其免疫机制的初步研究

观察钩端螺旋本ＤＮＡ疫苗的保护效果,并探讨疫苗的免疫机制。方法：应用定向克隆的方法构建ＤＮＡ疫苗ｐｃＤ－ｆｌａＢ,肌肉途径免疫豚鼠,观察豚鼠攻击感染钩体后保护率,以ＥＬＩＳＡ法检测特异性抗体ＩｇＧ工观察疫苗对豚鼠腹腔巨噬细胞分泌的ＴＮＦ活性的影响。结果该疫苗对同型钩体的保护率为１００％,特异性抗体水平在疫苗注射第６周达到高峰,ＴＮＦ的活性明显增高。结论：ＤＮＡ疫苗ｐｃＤ－ｆｌａＢ对同型钩体感染有较     

重组日本血吸虫亲环素A的原核表达及免疫保护作用的研究

目的 克隆、表达日本血吸虫亲环素A(SjCyPA)基因,并对重组蛋白的免疫保护效果进行实验室评价.方法 构建pET28-SjCyPA重组质粒,转化感受态大肠杆菌BL21/DE3.用IPTG诱导表达,经亲和层析纯化目的蛋白.用PBS、rSjCyPA分别免疫小鼠后进行日本血吸虫尾蚴攻击感染,观察其免疫保护效果.结果 成功表达了可溶性SjCyPA蛋白并得到纯化,经Western blot鉴定为血吸虫蛋白.rSjCyPA免疫小鼠产生60.10％的减虫率和43.42％的减卵率,HE染色及Masson染色揭示实验组肝脏虫卵肉芽肿及纤维化明显减轻.结论 rSjCyPA具有较好的抗血吸虫感染和抗血吸虫病的作用,是一个有前景的血吸虫疫苗候选分子.     

日本血吸虫基因组DNA的提纯与鉴定

本研究采用酚─氯仿法提纯了日本血吸虫基因组ＤＮＡ，并对基因组ＤＮＡ进行了定量分析及微电泳鉴定。结果表明，１００ｍｇ日本血吸虫成虫（湿重）可提纯出约８０μｇ的ＤＮＡ，其基因组ＤＮＡ的大小＞３１Ｋｂ。该法提取的血吸虫基因组ＤＮＡ较纯，适合于以后进行ＤＮＡ限制性核酸内切酶谱分析和Ｓｏｕｔｈｅｒｎｂｌｏｔ分析。     

日本血吸虫DNA疫苗的构建及其保护性免疫的研究

为探讨血吸虫ＤＮＡ疫苗保护性免疫效果,首先构建、鉴定和表达日本血吸虫ＤＮＡ疫苗（ｐＣＤ－Ｓｊ３２）。实验结果表明：ｐＣＤ－Ｓｊ３２免疫ＢＡＬＢ／Ｃ小鼠能诱导产生抗日本血吸虫感染免疫力,减虫率为３５．６％～４４．４％,减卵率为３９．４％～６９．０％;１００μｇＤＮＡ一次肌肉注射,免疫后８周攻击感染组的效果好;ＣＤ８＋Ｔ淋巴细胞、ＩＬ－２、ＴＮＦ和ＩＮＦ－γ可能在血吸虫病免疫功能调控中起重要作用;ｐＣＤ－Ｓｊ３２能诱导宿主产生高滴度特异性抗体,并在体外能介导巨噬细胞产生抗体依赖细胞介导的细胞毒性（ＡＤＣＣ）免疫效应。结果提示,ｐＣＤ－Ｓｊ３２有可能发展为新的预防血吸虫病的亚单位疫苗。     

Immune responses against Schistosoma japonicum after vaccinating mice with a multivalent DNA vaccine encoding integrated membrane protein Sj23 and cytokine interleukin-12

Background The vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum ( S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S.japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses.Methods The plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-γ and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM).Results The plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45. 53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-γ but decreases in IL-4.No significant differences in CD4^ and CD8^ subgroup ratios were observed after the challenges.Conclusions The multivalent DNA vaccine pVIVO2-1L12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Thl responses and, hence, the protective immunity.