猪带绦虫六钩蚴的体外培养

试验首次以人工胃液、人工肠液在离体的情况下将猪带绦虫的六钩蚴离体孵化与激活，并使用ＲＰＭＩ１６４０对激活的六钩蚴进行了体外培养。六钩蚴在培养的前几天内的变化十分明显，运动频率也较高。培养到第１０天时，可见到明显的二层体壁。     

猪带绦虫六钩蚴和未成熟期整尾蚴生化指标的变化

本试验检测了六钩蚴和猪体内发育至30日的囊尾蚴的18项生化指标，结果显示，在六钩蚴阶段有部分生化指标未能检测到，而在猪体内发育30日龄的囊尾蚴则全部检测到，提示在六钩蚴阶段虫体处于休眠状态态，由六钩蚴到未成熟期囊尾蚴是各个生化代谢通路的激活过程。     

豆状带绦虫六钩蚴的体外培养

(一)豆状带绦虫虫卵:采自以豆状囊尾蚴人工感染的家犬。 (二)RPMI1640培养基:美国GIBC公司生产。 (三)虫卵的预处理及六钩蚴的培养:将人工感染豆状带绦虫的犬杀死,取出绦虫、充分洗涤后将孕卵节片撕破、纱布过滤后离心、弃上清液,虫卵中加入0.02％洗必泰溶液室温下作用1小时,最后用灭菌生理盐水洗涤数次并配成一定浓度的虫卵悬液。取15万虫卵以2000r/m离心5分钟,弃上清液,沉淀物中加入人工胃液37℃水浴1小时后     

人工感染的亚洲带绦虫六钩蚴在幼猪体内的分布和致病观察

亚洲带绦虫是以猪为中间宿主的人体带绦虫,为了研究其幼虫的肝趋向性和组织选择性,试验分别用亚洲带绦虫虫卵灌胃和酶法孵化出六钩蚴后以静脉注射方式感染幼猪,35d后剖检,观察六钩蚴在猪体内的分布、发育和引起病变的特征。结果表明:两种途径入侵家猪的亚洲带绦虫六钩蚴都仅在肝脏发育成囊尾蚴;经静脉感染的幼猪肝脏、肺脏、肾脏、肌肉和皮下组织可检出以嗜酸性肉芽肿为主要特征的病变;经灌胃感染的幼猪则仅见肝脏出现病变,其他组织器官未见明显病变。     

猪带绦虫六钩蚴cDNA文库的构建与筛选

在体外将猪带绦虫虫卵孵化为有活力的六钩蚴。采用商品化试剂盒抽提六钩蚴总 RNA、m RNA,反转录为双链c DNA,再加 Eco R Adapter。在去除小片段后 ,dsc DNA与 λgt11噬菌体 DNA连接 ,经蛋白包装 ,构建了猪带绦虫六钩蚴λgt11c DNA文库。该文库效价为 3.5× 10 9pfu/ m L ,重组 DNA片段大小为 0 .5～ 3.2 kb,平均为 1.6 kb,蓝白斑比 1∶ 9。用抗体探针对文库进行免疫学筛选 ,在消除非特异性反应的基础上筛选约 10 6 重组子 ,共得到 118株强阳性克隆 ;应用 PCR鉴定上述部分阳性克隆 ,均扩增到 0 .5 kb以上的片段。结果显示 ,构建的文库合格 ,含六钩蚴所有抗原基因 ,可用于六钩蚴 c DNA克隆的筛选 ;用免疫学与 PCR联合筛选 c DNA文库 ,可消除假阳性     

猪带绦虫六钩蚴TSOL16基因的克隆与序列分析

利用RT-PCR技术,从猪带绦虫六钩蚴中成功地克隆了TSOL16基因。序列分析表明,猪带绦虫六钩蚴TSOL16基因的开放阅读框（ORF）为402 bp,编码134个氨基酸,与报道的T.ovisTO16基因同源性为95.9%,推导的氨基酸序列同源性为92.6%。同时利用生物信息学和分子生物学软件对TSOL16基因编码的蛋白进行结构预测。结果表明该蛋白为一结构松散的球状蛋白。     

体外人工肠液激活与未激活的猪带绦虫六钩蚴差异蛋白质组学比较

为建立一种用于比较猪带绦虫六钩蚴(Taenia solium)激活前后蛋白差异表达的二维液相色谱方法,本研究对人工肠液激活前后的六钩蚴进行一维色谱聚焦和二维反相色谱分析,以0.3pH为单位在pH7.71～pH5.31的范围内梯度分离出208个激活和197个未激活的六钩蚴蛋白.Mapping tools软件分析结果表明,六钩蚴激活前后的差异蛋白数量为77个,其中激活的六钩蚴差异蛋白44个,未激活的六钩蚴差异蛋白33个.该方法具有分辨率高、重现性好、自动化程度高的特点,为研究六钩蚴与其宿主间的寄生机制提供研究方法.     

猪带绦虫六钩蚴免疫原基因的克隆、表达与结构预测

从猪带绦虫六钩蚴cDNA表达文库中筛选、克隆的Ts01基因，设计表达性引物，亚克隆到pGEX-4T-1表达载体，构建了pGEX-GST-Ts01的融合表达重组载体，转化BL21寄主菌和IPTG诱导表达后，经SDS-PAGE和Western蛋白分析．表明成功地高效表达了约40000右的融合蛋白质．与预测的相对分子质量大小一致，表达量达40％左右。表达产物具有免疫学活性，而且呈可溶性，未形成包涵体，这与用蛋白质软件分析的Ts01编码蛋白水溶性相一致。     

以包涵体形式表达的猪带绦虫六钩蚴重组原的复性与纯化

应用比浊法,可溶必蛋白百分比,夹心ＥＬＩＳＡ及变性与非变性ＳＤＳ－ＰＡＧＥ等方法来评价猪带绦虫六钩蚴重组抗原的不同复性条件。透析法复性,透析液为含２ｍｏｌ／Ｌ尿素,１６０℃ｍｏｌ／ＬＰＥＧ４０００,１．０ｍｍｏｌ／Ｌ－０．１ｍｍｏｌ／ＬＧＳＨ－ＧＳＳＧ的ＰＨ９．０的Ｔｒｉｓ－ＨＣｌ缓冲液,蛋白浓度为２－５ｍｇ＝ｍＬ,以静置缓慢透年为佳,上述条件任意缺一或快速透析,则免疫活性下降,二聚体,多聚体明显     

猪带绦虫六钩蚴TSOL16基因的表达及免疫原性分析

本研究采用RT-PCR技术扩增猪带绦虫六钩蚴TSOL16免疫原基因,将扩增产物与pMD18-T载体连接,重组质粒经PCR、酶切鉴定后进行测序;构建TSOL16基因的pGEX-4T-1原核表达载体,用IPTG诱导表达后进行SDS-PAGE、Western blot分析;将所表达的蛋白免疫小鼠,经ELISA检测血清抗体验证其免疫原性。结果显示:所克隆的TSOL16基因片段长度为559bp,含有1个402bp的开放阅读框架,编码134个氨基酸,与已报道的猪带绦虫六钩蚴TSOL16基因核苷酸序列同源性为99%;表达的融合蛋白大小为40Ku,并能被猪带绦虫六钩蚴阳性血清所识别;免疫小鼠在1周后即检测到血清抗体,第30d即达到较高水平,说明该融合蛋白具有较好的免疫原性。     

Infection of normal C3H/HeN mice with Taenia saginata asiatica oncospheres

Normal C3H/HeN female mice were used to develop an animal model of Taenia saginata asiatica oncosphere infection. The host cellular immune response in this model was analyzed by a cytokine enzyme-linked immunosorbent assay (cytokine ELISA) and flow cytometry. Tumor-like cysts containing cysticerci were recovered from the inoculation sites of female mice 7 weeks postinfection with the T. saginata asiatica oncospheres. A sharp increase and sustained elevation in the ability of spleen cells to produce interferon-γ and interleukin (IL)-2 revealed that cellular immunity played an important role during the infection. An immediate increase in the levels of IL-6 at 1 week postinfection indicated the induction of a local acute inflammatory response. However, no significant change in the levels of IL-10 indicated that Th2 cells were not involved in this immune response. The patterns of cell distribution revealed by flow cytometry also supported the same finding. These results suggested that Th1 cells played a major role in the immune response in C3H/HeN mice during the early stages of the oncosphere infection and that the Th2 response was not induced during the stage of cysticercus formation.     

Specific and shared antigenic components of Taenia saginata oncospheres

Taenia saginata oncosphere components were analysed by double diffusion. Antigenic components in saline or detergent (Triton x-100) extracts of T saginata oncospheres were identified using a rabbit polyclonal serum directed against the oncosphere and compared with extracts prepared from the metacestodes and proglottids of T saginata and six other helminths commonly found in cattle. There were seven antigenic components found in the saline extract of the oncospheres, of which six were shared with the metacestodes and proglottids. None of these components was consistently different from those of the other six helminths. However, one of the four components in the detergent extract of the oncospheres was present in neither of the extracts of other stages of T saginata nor in extracts of other helminths. This may be of diagnostic significance.     

Effect of Paecilomyces lilacinus on the viability of oncospheres of Taenia hydatigena

Paecilomyces lilacinus (Thom) Samson is a saprophytic hyphomycete from the soil with biological activity on helminth eggs. We evaluated the influence in vitro of P. lilacinus on the viability of the oncospheres from Taenia hydatigena, a parasite cestode of dogs and sheep. The eggs were exposed to the fungus strain in sterile distilled water and observed by light microscopy at days 4, 7 and 14 post-inoculation, and the viability was evaluated. The viability found in the exposed P. lilacinus oncospheres was significantly different in all observations. P. lilacinus exercised a negative biological activity on T. hydatigena eggs in vitro.     

氧化应激对断奶仔猪组织抗氧化酶活性和病理学变化的影响

采用单因子试验设计,探讨用5%氧化鱼油(过氧化值为786.50 meqO2/kg)和腹腔注射12 mg/kg敌快死(diquat)对断奶仔猪肝脏抗氧化酶活性和组织病理学变化的影响.试验共分3个处理,每个处理8个重复,每个重复1头猪.结果发现,与新鲜鱼油组相比.氧化鱼油和diquat处理降低了试验猪肝脏抗氧化酶活性和提高了MDA含量,降低了空肠绒毛高度和增加了隐窝深度,空肠黏膜和肝脏发生不同程度的病理变化.结果表明,氧化鱼油和diquat都可以对断奶仔猪造成不同程度的氧化应激,相对于氧化鱼油的氧化应激程度而言,由diquat诱导的氧化应激程度更强一些.     

猪繁殖与呼吸综合征病毒SCQ株的仔猪回归试验及病毒在体内的分布研究

猪繁殖与呼吸综合征（PRRS）是由猪繁殖与呼吸综合征病毒（PRRSV）引起的猪的一种新的传染性疾病。该病自发现以来已经给各主要养猪国家带来了巨大的经济损列。1998年我们从重庆地区发病猪场的母猪血清中分离到PRRSV—SCQ毒株，经鉴定属美洲型毒株。因为用分离株进行病理机制研究方面的报道则更少。本试验以PRRSV重庆分离株人工感染易感仔猪，以复制出PRRS病症，研究病理变化特征和确定病毒抗原在仔猪体内的定植情况，为临床诊断和有效预防该病提供理论参考，也为进一步确证分离到的SCQ毒株属PRRSV提供佐证。     

Protein synthesis changes in the liver of piglets infected with Strongyloides ransomi

In piglets infected with Strongyloides ransomi, the incorporation of ¹⁴C-L-amino acid into liver protein was increased on days 7 and 14 post inoculation (PI), while on day 21 PI it was distinctly below the initial values.There was an increase of reduced glutathione (GSH) on day 7 PI and a decrease on day 14 PI. At day 21 PI the values were still below those of the controls.The concentration of plasma Cl, Na, K, Ca, Mg, Fe, Cu and Zn decreased from day 13 PI. The plasma levels of Na, K, Fe and chloride returned to normal on day 23 or 29 PI, but the other electrolytes had not regained their normal values at the end of the experiment (29 day PI).There was no change in electrolyte concentrations in heart, kidney and skeletal muscle. In the liver the concentrations of Ca, Fe and Cu increased. In the spleen the contents of Ca and Cu increased, whereas the fat content distinctly decreased.The enzymes GOT, GPT, LDH, LAP, ICDH, ALD, AP, SP and γ-GT were measured in blood plasma. Only alkaline phosphatase (AP) decreased markedly.     

豆状带绦虫六钩蚴Tp-dUTPase基因的表达及血清学诊断效果

采用PCR方法从豆状带绦虫六钩蚴中扩增得到Tp-dUTPase基因编码区,构建了pET32a-Tp-dUTPase原核表达载体,以重组蛋白dUTPase作为包被抗原建立的兔豆状囊尾蚴病Dot-ELISA诊断方法对226份兔血清进行了检测。结果显示,Tp-dUTPase基因编码区长447bp,编码148个氨基酸,表达的重组蛋白相对分子质量为36 200,该重组蛋白能与家兔豆状囊尾蚴病阳性血清特异性结合。以重组蛋白Tp-dUTPase作为包被抗原建立的Dot-ELISA诊断方法显示有较高的特异性（85.7%~92.9%）和敏感性（86.4%~88.0%）,与其他种类兔寄生虫病阳性血清有较低的交叉反应。结果表明,纯化的Tp-dUTPase重组蛋白可作为兔豆状囊尾蚴病的诊断抗原。