日本血吸虫重组蛋白TMEM66P的表达和多克隆抗体的制备

血吸虫病是由血吸虫尾蚴感染引起的一种人畜共患寄生虫病,对宿主造成严重病理性损害,是WHO确定的六大重点热带病之一。血吸虫体被直接与宿主的免疫系统相接触,是宿主与虫体信息交流的重要界面,位于虫体体被跨膜蛋白的功能研究尤显重要。本文对日本血吸虫膜蛋白(TEME66P)的cDNA进行表达和抗血清的制备。首先,利用生物信息学分析TMEM66P跨膜结构,并构建系统进化树,构建重组原核表达质粒并表达纯化重组蛋白,制备抗血清,通过Western blot对抗血清的特异性进行检测。研究表明TMEM66P存在跨膜区,其氨基酸序列与曼氏血吸虫同源性为67.79%,抗血清能够识别出全虫蛋白中TMEM66P,具有良好的特异性。本研究为进一步探索TMEM66P在日本血吸虫中的功能奠定了初步基础。     

尾蚴抗原特异性的T细胞克隆的建立及其功能测定——Ⅱ.T细胞克隆的功能测定

从早期感染曼氏血吸虫的C_(57)BL/6小鼠纵隔淋巴结细胞建立七株T细胞克隆。H~3-标记的淋巴细胞转化实验显示可溶性曼氏血吸虫尾蚴抗原、ConA和重组的人IL-2可促进这些T克隆细胞增殖,可溶性曼氏血吸虫虫卵抗原、成虫抗原及非特异性抗原PPD、纯化的老鼠IL-4均不起作用;体外实验显示T克隆细胞可诱导和增强对血吸虫尾蚴抗原特异性的肉芽肿反应;体内实验证实它们可调节血吸虫尾蚴抗原特异性的迟发型超敏反应。本文的结果为加深理解血吸虫病的免疫机制提供了一些实验依据。     

IgM单抗体内,外杀伤日本血吸虫的研究

本文报道应用急性感染日本血吸虫的Ｂａ１ｂ／ｃ小鼠脾细胞和ＳＰ２／０骨髓瘤细胞融合，获得了一株具有抗血吸虫攻击感染保护作用的ＩｇＭ单抗ｓｓｊ１４。在被动免疫转移试验中，这株单抗在小鼠体内提供了２７．６９％～６８．５％的减虫率。体外ＡＤＣＣ试验显示该单抗参与了巨噬细胞和嗜酸性粒细胞介导的杀伤血吸虫童虫的作用，免疫化学特性测定表明该单抗同时对血吸虫尾蚴、成虫和虫卵三期虫体抗原起反应，靶抗原的分子量为２９０ＫＤ，分别位于血吸虫童虫和成虫的表膜及虫卵的卵壳。靶抗原表位的化学性质为多糖类，它同时被辐照致弱尾蚴免疫的大鼠血清、血吸虫慢性感染人血清和小鼠血清所识别。本文结果表明，ＩｇＭ抗体和多糖类抗原在血吸虫病免疫保护作用中发挥了重要的作用。     

日本血吸虫信号转导蛋白sjwnt10a基因的克隆及其在童虫和成虫中mRNA表达量的变化

利用RACE技术从19日龄的日本血吸虫童虫中扩增了1个Wnt家族基因，并对其进行了生物信息学分析。同源性分析表明，该基因为日本血吸虫新基因，完整开放阅读框（ORF）长1896bp，编码631个氨基酸，理论分子质量为73．3ku。该基因编码的氨基酸序列具有wnt家族蛋白的典型特征，与人、鼠Wnt10a的氨基酸序列同源性均为26％，推测为血吸虫的Wnt10a基因，命名为Sjwnt10a（GenBanK登录号DQ643829）。利用实时荧光定量PCR分析该基因在日本血吸虫不同发育阶段虫体中的表达情况，结果显示该基因在19日龄的日本血吸虫童虫中表达量最高，在14日龄童虫和44日龄雄虫中也有表达，但分别仅为19日龄童虫表达量的8．8％和5．0％，而在31日龄成虫和44日龄雌虫中没有检测到该基因。结果提示，童虫差异表达的Sjwnt10a基因可能对童虫的生长、发育至关重要。     

东方田鼠肝脏裂解物筛选日本血吸虫成虫噬菌体展示cDNA文库

东方田鼠对血吸虫具有天然抗性。为寻找东方田鼠抗血吸虫抗性相关靶基因，探索其分子机理，我们将日本血吸虫童虫与东方田鼠不同组织／器官裂解物共培养，比较它们对血吸虫童虫的杀伤效果，之后，以杀伤效果较好的肝脏裂解物为探针，以亲和淘洗方法筛选日本血吸虫成虫（44d）噬菌体展示cDNA文库。经三轮筛选后，随机挑取92个噬菌体克隆进行序列测定及生物信息学分析，获得了19个有效EST序列，其中15条EST序列与已知基因或表达序列标签同源，4个EST序列与已知基因或表达序列标签均无同源性，为新的表达序列标签。对19个有效EST编码蛋白的功能预测结果显示，其中6个为卵壳蛋白（Eggshell protein）相关基因，10个EST及其编码蛋白的功能与血吸虫基因表达调控及蛋白质加工修饰相关。研究结果提示，东方田鼠直接或间接地影响血吸虫基因表达，进而影响血吸虫的发育，可能是东方田鼠具有抗血吸虫特性的一个重要分子机理。     

血吸虫期别差异表达功能基因的研究进展

血吸虫的生长发育过程中,其抗原表 达呈现明显的期别差异性。利用分子生物学 技术,从期别差异表达的功能基因中筛选有 意义的功能蛋白,并深入研究它们的生物学 活性,是近年来血吸虫基因组研究的一个热 点,这对开发新的血吸虫疫苗和寻找药物靶 标,对防治血吸虫病具有重要意义。文章对 血吸虫虫卵、尾蚴、童虫、成虫各个阶段的特 点及这些阶段差异性表达的功能基因研究进 展作了综述,并简要介绍了一些在血吸虫差 异表达基因克隆中运用的生物学技术。     

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。     

东方田鼠重复感染日本血吸虫试验研究初步

本试验以1000条日本血吸虫尾蚴同时攻击感染东方田鼠洞庭湖种群和东方田鼠宁夏指名亚种进行重复感染对比试验，并在攻击后不同天数分别同时解剖相数目的两种群东方田鼠，收集其体内不同时段，不同部位的日本血吸虫童虫，比较结果证实东方田鼠对日本血吸虫具有天然的抗性，其抗性是可遗传的，不以东方田鼠的地域分布不同、生活环境的差异或传代数目的变化而有所不同，与获得必免疫无关。     

日本血吸虫童虫冷冻保藏复苏后生活史循环的研究

本文报告了日本血吸虫童虫经液氮冻藏复苏后的生活史循环的情况。童虫冻藏３６天和５０天后腹腔注射感染小鼠，亲代成虫回收率为９．０和８．９％。感染鼠肝内的子代虫卵孵出的子１代毛蚴感染钉螺，感染率为５９和６７％；平均每个钉螺第１次逸出的子１代尾蚴数为８００～９００条。子１代尾蚴感染小鼠后，其子１代成虫回收率为５５和５６％。合抱雌、雄成虫的体长、性腺的发育和虫体对宿主的致病力与正常途径感染者无明显差异。     

单性血吸虫带虫免疫对继发血吸虫病的影响

研究表明,辐照致弱血吸虫尾蚴疫苗能够诱导宿主产生较高的免疫保护作用,是所有血吸虫候选疫苗中保护力最强的,且辐照致弱尾蚴在宿主体内不能发育成熟,不会对宿主造成严重的病理损害。单性感染的血吸虫同样不能在宿主体内发育成熟,其发育状态停留在童虫阶段,也不会对宿主造成严重的病理损害,在童虫状态下对宿主持续进行的免疫刺激,是否也能诱导宿主对继发血吸虫病产生较高的免疫保护作用呢？为此,本研究通过让宿主携带单性血吸虫雄虫的带虫免疫方式来检测其对继发血吸虫病的影响。选用雄性血吸虫尾蚴感染BALB/c小鼠（100条/只）,于单性血吸虫尾蚴攻击后第5、7 w用吡喹酮进行治疗（300mg/kg）,第9 w进行雌雄血吸虫混合尾蚴攻击实验动物（40条/只）,第15 w剖杀实验动物,收集宿主荷虫、宿主肝卵负荷、肝卵孵化、宿主肝单卵肉芽肿体积数据,并检测宿主血清特异性抗体变化趋势。结果表明单性带虫免疫可以引起宿主肝虫卵孵化率及组织病理损伤显著降低,对宿主有一定的免疫保护效果。     

Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica

Immature Fasciola hepatica release a papain or cathepsin B-like proteolytic enzyme which cleaves immunoglobulins (Ig) of mouse, rat, rabbit and sheep in vitro. Mouse IgG and IgM molecules are both susceptible to cleavage as is hemoglobin. Whether single or multiple proteases are responsible for Ig cleavage is unknown. The proteolytic activity of secreted enzyme(s) is optimal at pH 3.5-4.5, but activity is also present at pH 7. Proteolysis is enhanced in the presence of 5 mM dithiothreitol or 100 mM cysteine. Based on studies with protease inhibitors, the F. hepatica enzyme activity has been identified as a thiol protease. It is destroyed by heating at 56 degrees C for 1 h, but retains activity after storage at -20 degrees C for 7 days. Whether inhibition of the proteolytic activity increases the susceptibility of F. hepatica immature worm to any extant immune effector mechanisms in hosts remains to be determined.     

饲料锌含量对方格星虫稚虫生长性能、体成分、体腔液中锌含量及碱性磷酸酶活性的影响

以蛋氨酸锌为锌源,在等氮等能的基础饲料中添加不同水平的锌,配制实际锌含量分别为9.3、31.7、49.9、90.1、168.6和326.5 mg/kg的6种试验饲料,饲喂平均体重为(14.54±0.10)mg的方格星虫稚虫8周,用以研究饲料锌含量对方格星虫稚虫生长、体成分、体腔液中锌含量及碱性磷酸酶活性的影响。每种试验饲料设3个重复,每个重复饲喂400尾方格星虫稚虫。结果表明:饲料锌含量对方格星虫的成活率、增重率和特定生长率均有显著影响(P0.05),其中增重率和特定生长率均在饲料锌含量为49.9 mg/kg时达到最高值,并显著高于饲料锌含量最低(9.3 mg/kg)和最高(326.5 mg/kg)的组(P0.05)。饲料锌含量对虫体粗脂肪和粗灰分含量有显著影响(P0.05),虫体粗脂肪含量随着饲料锌含量的提高呈先增后降的趋势,而粗灰分含量的趋势则相反。饲料锌含量对虫体水分和粗蛋白质含量没有显著影响(P0.05)。饲料锌含量对虫体体腔液锌含量和碱性磷酸酶活性均有显著影响(P0.05)。虫体体腔液锌含量随着饲料锌含量的提高而不断上升,在饲料锌含量为326.5 mg/kg时达到最高值;而虫体体腔液中碱性磷酸酶活性则随着饲料锌含量的提高先上升后趋于稳定,当饲料锌含量为49.9 mg/kg时达到最高值。以增重率为评价指标,通过回归分析得出方格星虫稚虫饲料中适宜锌含量为41.93 mg/kg。     

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.     

Characterization of a novel vaccine candidate and serine proteinase inhibitor from Schistosoma japonicum (Sj serpin)

Serine proteinase inhibitors (serpins) represent an important superfamily of endogenous inhibitors that regulate proteolytic events active in a variety of physiological functions. Immunological screening of a Schistosoma japonicum adult worm cDNA expression library with sera of Microtus fortis, a naturally resistant vertebrate host, has identified one clone that encoded for a sequence homologous to those of the serpin superfamily. The full-length sequence encoding S. japonicum serpin (Sj serpin) was amplified from adult worm cDNA by using 5'-RACE-PCR and subsequently cloned into the prokaryotic expression vector pET28c. The full-length Sj serpin fusion-protein with his-tag was expressed in E. coli, purified by affinity chromatography and used to immunize New Zealand white rabbits. Sj serpin is located on the tegument in S. japonicum adult worms. C57BL/6 mice immunized with Sj serpin induced the production of high levels of specific IgE and IgG1 subclass antibodies as well as a marked IL-4 response. Lymphocyte surface marker analysis revealed proliferation of CD19 expressing B cells, indicating a predominant Th2-type response to Sj serpin. Immunized mice developed moderate protection against infection of S. japonicum as demonstrated by a 36 and 39% reduction in the recovery of adult worms and eggs, respectively. These data suggested a role for Sj serpin as a vaccine candidate or as a novel target for anti-schistosome drugs.     

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。     

Chemotactic activity of hemocytes derived from a brackish-water clam, Corbicula japonica, to Vibrio parahaemolyticus and Escherichia coli strains.

Hemocytes from adult and juvenile specimens of a brackish-water clam, Corbicula japonica, were attracted chemotactically to live cells of Vibrio parahaemolyticus and Escherichia coli strains in a balanced salt solution, which was enhanced significantly in the presence of respective C. japonica plasma. Chemotactic attractions of adult's and juvenile's hemocytes were seen also in artificial seawater at a similar level to those in the balanced salt solution. Chemotactic attractions of juvenile's hemocytes to these strains were lower in level than those of adult's hemocytes. C. japonica plasma seems to facilitate for C. japonica hemocytes to recognize these organisms.     

Proteolytic enzymes from Trichinella spiralis larvae.

Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytic enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containing gelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.     

添加槐花粉对白三叶青贮发酵品质的影响

采用单因子随机设计,研究添加不同比例的槐(Sophora japonica)花粉对白三叶(Trifolium repens)青贮发酵品质的影响,以期为槐花应用于青贮饲料生产提供科学依据.分别设置白三叶单独青贮(C)、95％白三叶+5％槐花粉青贮(T1)、90％白三叶+10％槐花粉青贮(T2)、85％白三叶+15％槐花粉...     

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.