应用cDNA微阵列芯片技术筛选猪蛔虫性别差异表达基因的研究

采用cDNA微阵列芯片技术，从所构建的猪蛔虫雌、雄成虫cDNA消减文库分别挑取1044和1119个克隆，PCR扩增其插入片段，经纯化后点样于预先处理好的基片上（双点杂交），制备成cDNA微阵列芯片。将分别标记荧光素Cy3-dUTP和Cy5-dUTP的雌虫和雄虫cDNA探针，与制备好的cDNA芯片杂交（平行进行反标杂交试验）。根据每个点杂交后的Ratio值，筛选出双点杂交和正反标中都同时具有表达差异的基因克隆共1559个。将表达差异最明显的前831个克隆进行测序，获得720个有效序列，经生物信息学分析发现，雄虫特异表达的主要精于蛋白和雌虫特异表达的卵巢信息蛋白的基因序列多数与新杆属线虫存在同源性，有31个可能是新的ESTs。性别差异表达基因及其相关生物信息的获得为下一步研究基因功能奠定了基础。     

猪蛔虫感染相关基因28A02的表达谱及其全长cDNA的克隆和分析

为鉴定猪蛔虫与感染相关的差异表达基因,本实验由猪蛔虫感染期幼虫差异消减cDNA文库中挑选EST序列(28A02)为研究对象,首先,经半定量RT-PCR方法分析该基因在各期虫体的表达情况。结果显示:该基因在猪蛔虫感染期幼虫和肺三期幼虫中均有表达,并且在感染期幼虫表达丰度最高,在其它各期虫体包括雌虫、雄虫、虫卵和第四期幼虫均未检测出该基因的表达。其次,以已知的EST序列为模板,采用RACE技术获得了864 bp的全长cDNA序列,包含一个完整的长约450 bp的开发阅读框。另外,经生物信息学分析显示,该基因编码的氨基酸序列与众多物种的ATP合酶有43%～47%的相似性,推测该基因可能参与编码猪蛔虫的线粒体ATP合酶,这为进一步研究其功能奠定了基础。     

猪蛔虫不同发育阶段09G10基因的表达分析

为进一步鉴定和分析猪蛔虫感染相关基因,从构建的猪蛔虫感染期幼虫消减cDNA文库中筛选出一基因(EST编号为09G10),以肌动蛋白(β-actin)为内参,采用半定量RT-PCR方法分析该基因在猪蛔虫不同发育阶段的表达情况。通过09G10基因与内参β-actin基因的积分光密度的比值结果显示,09G10在感染期幼虫中呈现高丰度表达,在肺第3期幼虫和第4期幼虫期有微量表达,在其他各期虫体包括肝第3期幼虫、雌虫、雄虫和虫卵均未检测出该基因的表达量,表明09G10基因可能是猪蛔虫幼虫期所特有的基因,在幼虫感染宿主过程中可能扮演重要角色,为进一步研究该基因的功能提供了依据。     

猪蛔虫感染期幼虫抑制消减cDNA文库的构建

为筛选与线虫感染性相关的基因,本研究以猪蛔虫为对象,构建猪蛔虫感染期幼虫差异表达消减cDNA文库,为研究线虫期特异性发育的分子机制奠定基础。分别提取感染期幼虫和其它各期幼虫及成虫的总RNA,纯化mRNA后,采用Clontech公司PCR-selectTM试剂盒进行反转录合成cDNA并进行抑制消减杂交(SSH),构建猪蛔虫感染期幼虫差异表达的消减cDNA文库,并采用Southern斑点杂交进行消减效率的检测。随机从文库中抽取45个克隆进行测序及在线BLAST分析。试验结果表明,感染期幼虫差异表达的消减cDNA文库具有较强的特异性;在得到的41个表达序列标签(ESTs)中,有40个ESTs与已报道的基因有较高的相似性,主要代表猪蛔虫第三期幼虫基因和成虫头部基因,有1个cDNA片段可能代表新基因。猪蛔虫感染期幼虫差异表达消减cDNA文库的成功构建,为进一步研究幼虫发育差异表达基因的功能奠定了基础。     

猪蛔虫微管蛋白α-链基因(13E09)在不同发育期的mRNA表达研究

猪蛔虫病严重危害我国养猪业的发展,通过研究在感染期的表达基因来研究其发育繁殖机制,以达到预防和控制该病的目的已成为猪蛔虫研究的发展趋势。本研究以β-actin为内参,采用半定量RT-PCR方法鉴定分析猪蛔虫微管蛋白α-链基因(13E09)丰度随不同阶段虫体在虫体不同发育阶段的mRNA表达水平的变化,结果显示,该在感染期幼虫呈高丰度表达外,肠四期即(L4)幼虫也有多量表达,在雄虫、虫卵期也有少量表达,但在雌虫和肺三期幼虫均未检测出该基因的表达,该基因是在感染期幼虫呈高丰度表达的基因,可能在感染宿主过程中起重要作用。这为进一步研究该基因的功能提供了科学依据。     

犬弓首蛔虫卵黄原蛋白基因的克隆及表达分析

为克隆犬弓首蛔虫(T.canis)的卵黄原蛋白(YP)基因,本研究根据T.canis cDNA文库中的EST数据,采用RT-PCR方法扩增T.canis的YP基因(TcF-YP),并克隆至pGEM-T载体中进行测序。测序结果表明,TcF-YP基因片段大小为492 bp,编码163个氨基酸残基。同源分析显示,TcF-YP基因与猪蛔虫的卵黄原蛋白基因同源性最高,为78%。同时对该基因在T.canis雌虫、雄虫和雌虫各组织的表达谱进行研究,结果显示TcF-YP基因只在T.canis雌虫中高量表达,是雌虫特异的基因。对雌虫各组织的基因表达谱分析显示TcF-YP基因在子宫和肠中高量表达,在卵巢中有极微量的表达。本实验为TcF-YP基因的功能研究奠定了基础。     

捻转血矛线虫ES24抗原基因的克隆与表达特性分析

根据捻转血矛线虫ES24抗原基因序列(U64793.1)设计1对特异性引物,用RT-PCR方法扩增出大小约为670 bp的DNA片段。将该DNA片段克隆到pMD18-T载体后进行序列测定和分析,结果发现该基因与GenBank中已知的捻转血矛线虫24 ku ES抗原基因的相似性达96%～98%。将该基因的开放阅读框插入pET28a(+)载体中,获得原核表达质粒pET28/ES24,并转化大肠杆菌BL21。重组细菌用IPTG诱导,经SDS-PAGE分析,结果表明该基因获得了表达,重组蛋白分子量大小约为25 ku。用实时荧光定量PCR技术对该基因在捻转血矛线虫的虫卵、第3期幼虫、雌虫和雄虫等不同发育阶段、不同性别虫体内的表达情况进行了定量分析,结果显示ES24基因在雄性成虫中表达量最高,雌虫和虫卵其次,在第3期幼虫中表达最低。     

BmLSP基因启动子驱动DsRed在转基因家蚕中的表达分析

家蚕幼虫血清蛋白(BmLSP)是由脂肪体细胞合成的一种贮藏蛋白,是研究家蚕幼虫期发育调控的理想靶标。基于家蚕基因组数据,克隆了BmLSP基因5'端侧翼区～1.6 kb的调控序列,构建成以红色荧光蛋白基因DsRed为报告基因的pig-gyBac表达载体并进行转基因注射,在个体水平上验证该启动子的特性。结果表明:BmLSP-DsRed表达框以单拷贝形式整合至家蚕基因组;DsRed在转基因家蚕脂肪体中特异转录表达,其时期表达特征与BmLSP基因基本一致,随发育阶段的不同呈现有规律的变化,即幼虫期表达,但1龄、2龄眠期不表达,3龄、4龄眠期有微弱表达,上蔟后表达量逐渐降低直至化蛾阶段消失。提示BmLSP可能通过其启动子区的特异元件受激素的精确调控而行使功能。     

清平猪不同配套杂交组合生产性能比较试验

(1)应用28窝L♂×LW·Q♀三元杂交和26窝L♂×Q♀二元杂交,比较两者之间初生窝重、窝仔猪数、断奶窝重等繁殖性能,结果L♂×LW·Q♀三元杂交各项指标均高于L♂×Q♀二元杂交,分别高1.4kg、1.31头、9.2kg,初生窝重、窝仔猪数经统计分析P＜0.05,表明组间差异显著,断奶窝重经统计分析P＜0.01,表明组间差异极显著.这亦说明以清平猪为第一母本的三元杂交模式应成为清平猪产区主要的商品猪生产方式.(2)应用25头L♂×LW·Q♀三元杂交猪和21头L♂×Q♀二元杂交猪进行育肥饲养实验,L♂×LW·Q♀三元杂交猪平均日增重686g,L♂×Q♀二元杂交猪平均日增重613g,两者比较三元杂交猪比二元杂交猪平均日增重高73g,统计分析P＜0.01,表明组间差异极显著.L♂×LW·Q♀三元杂交猪与L♂×Q♀二元杂交猪的料重比比较,降低饲料消耗7.3%,育肥猪增重营养成本:DE下降7.8%,CP下降6.3%,说明L♂×LW·Q♀三元杂交猪在育肥性能上优于L♂×Q♀二元杂交猪.     

捻转血矛线虫重组磷脂酰肌醇转移蛋白对山羊外周血单个核细胞模式识别受体和细胞因子转录水平的影响

本文旨在研究捻转血矛线虫(Haemonchus contortus)磷脂酰肌醇转移蛋白(phosphatidylinositol transfer protein, HCPITP)对山羊的免疫调节功能。构建了原核表达重组质粒pET-28a-HCPITP并获得重组蛋白;分析HCPITP基因在虫卵、第三期幼虫、雌虫和雄虫中转录水平的变化;将重组蛋白与山羊外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)共孵育,研究重组蛋白对山羊PBMCs增殖、模式识别受体(pattern recognition receptors, PRRs)表达和细胞因子分泌的影响。结果显示,HCPITP基因在雌虫、雄虫、第三期幼虫和虫卵中相对转录水平分别为1、10.2、0.47和0.58。Western blot分析发现该重组蛋白具有较好的反应原性。该重组蛋白能够显著抑制ConA对PBMCs的增殖作用,重组HCPITP显著促进山羊PBMCs的IL-2、IL-4、IL-6和IL-17转录水平,主要促进模式识别受体TLR1与TLR10的转录。综上,HCPITP基因转录水...     

Susceptibility of fourth-stage Ascaris suum larvae to fenbendazole and to host response in the pig

Fenbendazole given at the rate of 2.5 g/kg of feed for 3 days had 100% efficacy against 4th-stage Ascaris suum larvae in 8 pigs. Eight control pigs had a total of 108 A suum. In 6 pigs infected 3 times with 3rd-stage A suum larvae and treated with fenbendazole after the larvae molted to the 4th stage, the challenge exposure-derived population was decreased by 64%. Similar sequential infections in 6 pigs similarly infected, but not treated with fenbendazole, decreased the challenge exposure-derived population by 98%; however, developing and/or adult worms from the vaccinating infections were present.     

Identification of larval-stage antigens of ascaris suum recognized with immune sera from pigs.

Antigens were identified from the third-stage larvae (L3) and lung-stage larvae of Ascaris suum by two-dimensional immunoblot method with antisera obtained from pigs that received chemically abbreviated Ascaris suum larval infections. Forty-seven and 13 spots were recognized as antigens from the L3 and lung-stage larvae, respectively. Their apparent molecular weight ranged from 20 to 101 kDa and their isoelectric point from 3.6 to 8.0. The present study provides a framework for further molecular cloning of those antigens and consequently leads to the development of recombinant peptide vaccines against A. suum.     

The effect of ivermectin treatment against inhibited early third stage, late third stage and fourth stage larvae and adult stages of the cyathostomes in Shetland ponies and spontaneous expulsion of these helminths.

A controlled and critical test on the efficacy of ivermectin against larval and adult stages of the cyathostomes was carried out in six yearling castrated male Shetland ponies. The ponies grazed together as one group from 3 May to 4 October 1990, after which they were housed. Three ponies were treated with ivermectin on 29 October while the others served as controls. The shedding of helminths in the faeces was followed in all ponies until necropsy on 14 November. Comparison of worm counts of both groups before and after necropsy showed no evidence for an effect of ivermectin against inhibited early third stage larvae (EL3) and mucosal late third stage (LL3) and fourth stage larvae (L4). However, a high, but not 100%, efficacy was observed against adults and lumenal L4. A remarkable observation was the high incidence of spontaneous expulsion of L4 and adult populations of some species in two of the untreated ponies.     

Immunization of pigs against Ascaris suum by sequential experimental infections terminated with fenbendazole during larval migration

Three experimental infections of weaning pigs with 2000 embryonated Ascaris suum eggs each, 11 days apart, followed each time by fenbendazole treatment, produced a significant host response when compared with similar infected or uninfected control pigs as assessed by response to a subsequent challenge with 100 embryonated A. suum eggs. The response elicited from pigs treated with fenbendazole on either 2, 3, and 4 days or, 6, 7, and 8 days after each experimental infection was expressed as a reduction in the number of pigs with A. suum, in the number of worms per pig, in the weight of male and female worms, and in the length of male and female worms. No differences in average daily weight gain, feed-conversion efficiency or histology of lungs and liver were noted among the 4 treatment groups.     

Comparative studies of experimental oral inoculation of pigs with Ascaris suum eggs or third stage larvae

This experimental study was designed to compare the acquired resistance in pigs to Ascaris suum eggs following 4-weekly oral immunizations with either 200 A. suum infective eggs or 50 A. suum third stage larvae (L3). The two immunized groups (n = 7) together with an unimmunized control group (n = 7) of pigs were challenged orally with 50 infective A. suum eggs per kilogram bodyweight on day 19 after the last immunization. Seven days post-challenge the group immunized with eggs showed signs of resistance as evidenced by reduced lung larval counts compared with the challenge control group. Such significant resistance was not observed in the L3-immunized group. However, a markedly increased inflammatory liver reaction and white spot formation was demonstrated in the L3-immunized pigs after challenge compared with both control animals and egg-immunized pigs. On the day of challenge only the egg-immunized pigs mounted an anti-Ascaris antibody response both in serum and in lung lavage fluid. Ascaris-antigen induced increased histamine release from peripheral leucocytes following both immunization and challenge could only be demonstrated in the egg-immunized pigs. On day 7 post-challenge local IgA-anti-Ascaris antibodies were further demonstrated in bile of the egg-immunized group and in the small intestine of both immunized groups. In conclusion, oral A. suum egg immunization of pigs induced a significant reduction in lung larval counts upon challenge. In contrast, oral L3 immunization seemed to prime the pigs as observed by the presence of stunted lung larval growth and increased liver reaction post-challenge with A. suum eggs.     

14株苏云金芽孢杆菌伴胞晶体蛋白对猪蛔虫第3期幼虫的毒性比较

14株苏云金芽孢杆菌经培养提取伴胞晶体蛋白并测定其蛋白含量。感染性蛔虫卵经胃感染小鼠 ,3d后宰杀并从小鼠肝脏内分离蛔虫第 3期幼虫 (L 3)。设置空白对照 ,将不同剂量的 Bt伴胞晶体蛋白与 L 3置于培养孔在 37℃的二氧化碳培养箱中 (含 5 % CO2 )培养 ,间隔 12 h检查幼虫活性 ,计算死亡率、校正死亡率及半数致死量。L D50 随着作用时间的延长而逐渐减小。14株细菌伴胞晶体蛋白对 L 3的毒力差异很大。 0 17株对 L 3的杀虫作用快速而持久、毒力最强 ,其 L D50 是所有 14个菌株中最小的 ,作用 12、2 4和 36 h时分别为 1.135 7、0 .2 32 2和 0 .15 32 g/ L。